Anti-pd-1 antibodies and methods of use thereof

ABSTRACT

The present invention provides antagonizing antibodies that bind to programmed cell death protein 1 (PD-1) and methods of using same. The anti-PD-1 antibodies can be used therapeutically alone or in combination with other therapeutics to treat cancer and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority, under 35 USC §119(e), to the followingUS provisional applications: U.S. Patent Application No. 62/089,658,filed Dec. 9, 2014, U.S. Patent Application No. 62/242,750, filed Oct.16, 2015, and U.S. Patent Application No. 62/251,973, filed Nov. 6,2015, each of which is hereby incorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “PC72121A_SeqListing_ST25.txt”created on Nov. 10, 2015 and having a size of 139 KB. The sequencelisting contained in this .txt file is part of the specification and isherein incorporated by reference in its entirety.

FIELD

The present invention relates to antibodies, e.g., full lengthantibodies that bind PD-1. The invention further relates to compositionscomprising antibodies to PD-1, and methods of using anti-PD-1 antibodiesas a medicament. Certain embodiments relate to methods of usinganti-PD-1 antibodies for the treatment, prevention and/or diagnosis ofvarious diseases, including hyperproliferative disease, such as cancer.

BACKGROUND

PD-1 is a 50-55 kDa type I transmembrane receptor that was originallyidentified in a T cell line undergoing activation-induced apoptosis.PD-1 is expressed on T cells, B cells, and macrophages. The ligands forPD-1 are the B7 family members PD-L1 (B7-H1) and PD-L2 (B7-DC).

PD-1 is a member of the immunoglobulin (Ig) superfamily that contains asingle Ig V-like domain in its extracellular region. The PD-1cytoplasmic domain contains two tyrosines, with the mostmembrane-proximal tyrosine (VAYEEL in mouse PD-1) located within an ITIM(immuno-receptor tyrosine-based inhibitory motif). The presence of anITIM on PD-1 indicates that this molecule functions to attenuate antigenreceptor signaling by recruitment of cytoplasmic phosphatases. Human andmurine PD-1 proteins share about 60% amino acid identity withconservation of four potential N-glycosylation sites, and residues thatdefine the Ig-V domain. The ITIM in the cytoplasmic region and theITIM-like motif surrounding the carboxy-terminal tyrosine are alsoconserved between human and murine orthologues.

Cancer immunotherapy has traditionally involved complicated methodsusing cells and individualized and time-consuming preparations.Recently, monoclonal antibody-based cancer immunotherapy based on theinterruption of suppressive signals that are delivered to the adaptiveimmune system has shown promise in the clinic within the setting ofoff-the-shelf systemic immunotherapy. However, there is a continuingneed in the art to obtain safer and more effective treatments forcancer.

SUMMARY

Antibodies that selectively interact with PD-1 are provided. It isdemonstrated that certain anti-PD-1 antibodies are effective in vivo toprevent and/or treat cancer. Advantageously, the anti-PD-1 antibodiesprovided herein bind human, cynomolgous monkey, and mouse PD-1. Alsoadvantageously, the anti-PD-1 antibodies provided herein are effectivein vivo to stimulate T cell proliferation.

Isolated antagonist antibodies that specifically bind to PD-1 andprevent or reduce the biological effect of PD-1 are provided herein. Insome embodiments, the antagonist antibody can be, for example, a human,humanized, or chimeric antibody. The invention disclosed herein isdirected to antibodies that bind to PD-1.

In one aspect, the invention provides an isolated antagonist antibodywhich specifically binds to PD-1, wherein the antibody comprises a heavychain variable region (VH) comprising a VH complementarity determiningregion one (CDR1), VH CDR2, and VH CDR3 of the VH having an amino acidsequence selected group the group consisting of SEQ ID NO: 3, SEQ ID NO:4; SEQ ID NO: 5; and SEQ ID NO: 6; and a light chain variable region(VL) comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL having anamino acid sequence selected from the group consisting of SEQ ID NO: 2;SEQ ID NO:7; SEQ ID NO: 8; and SEQ ID NO: 9.

In some embodiments, the VH region comprises the amino acid sequenceshown in SEQ ID NO: 3, 4, 5, or 6, or a variant with one or severalconservative amino acid substitutions in residues that are not within aCDR and/or the VL region comprises the amino acid sequence shown in SEQID NO: 2, 7, 8, or 9, or a variant thereof with one or several aminoacid substitutions in amino acids that are not within a CDR. In someembodiments, the antibody comprises a light chain comprising thesequence shown in SEQ ID NO: 39 and/or a heavy chain comprising thesequence shown in SEQ ID NO: 29 or 38. In some embodiments, the antibodycomprises a VH region produced by the expression vector with ATCCAccession No. PTA-121183. In some embodiments, the antibody comprises aVL region produced by the expression vector with ATCC Accession No.PTA-121182.

In another aspect, the invention provides an isolated antibody whichspecifically binds to PD-1, wherein the antibody comprises a VH CDR1comprising the amino acid sequence of SEQ ID NO: 13, 14, or 15, a VHCDR2 comprising the amino acid sequence of SEQ ID NO: 16, 17, 24, 25,27, 28, 35, or 36, a VH CDR3 comprising the amino acid sequence shown inSEQ ID NO: 18, 23, 26, or 37, a VL CDR1 comprising the amino acidsequence shown in SEQ ID NO:10, 22, 30, or 32, a VL CDR2 comprising theamino acid sequence shown in SEQ ID NO: 11, 20, or 33 and a VL CDR3comprising the amino acid sequence shown in SEQ ID NO: 12, 21, 31, or34.

In some embodiments, the antibody can be a human antibody, a humanizedantibody, or a chimeric antibody. In some embodiments, the antibody is amonoclonal antibody.

In some embodiments, the antibody comprises a constant region. In someembodiments, the antibody is of the human IgG₁, IgG₂, IgG_(2Δa), IgG₃,IgG₄, IgG_(4Δb), IgG_(4Δc), IgG₄ S228P, IgG_(4Δb) S228P, and IgG_(4Δc)S228P subclass. In some embodiments, the antibody is of the IgG4 isotypeand comprises a stabilized hinge, e.g., S228P.

In another aspect, the invention provides an isolated antibody whichspecifically binds to PD-1 and competes with and/or binds to the samePD-1 epitope as the antibodies as described herein.

In some embodiments, an anti-PD-1 antibody provided herein promotes IFNγand/or TNF secretion from T cells.

In some embodiments, an anti-PD-1 antibody provided herein promotesproliferation of T cells.

In some embodiments, an anti-PD-1 antibody provided herein inhibitstumor growth.

In some embodiments, an anti-PD-1 antibody provided herein binds humanPD-1 and mouse PD-1.

In another aspect, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of a PD-1 antibody asdescribed herein and a pharmaceutically acceptable carrier.

In another aspect, the invention provides an isolated polynucleotidecomprising a nucleotide sequence encoding a PD-1 antibody as describedherein. In another aspect, the invention provides a vector comprisingthe polynucleotide.

In another aspect, the invention provides an isolated host cell thatrecombinantly produces a PD-1 antibody as described herein.

In another aspect, the invention provides a method of producing ananti-PD-1 antagonist antibody, the method comprising: culturing a cellline that recombinantly produces the antibody as described herein underconditions wherein the antibody is produced; and recovering theantibody.

In another aspect, the invention provides a method of producing ananti-PD-1 antagonist antibody, the method comprising: culturing a cellline comprising nucleic acid encoding an antibody comprising a heavychain comprising the amino acid sequence shown in SEQ ID NO: 29 or 38and a light chain comprising the amino acid sequence shown in SEQ ID NO:39 under conditions wherein the antibody is produced; and recovering theantibody.

In some embodiments, the heavy and light chains are encoded on separatevectors. In other embodiments, heavy and light chains are encoded on thesame vector.

In another aspect, the invention provides a method for treating acondition in a subject comprising administering to the subject in needthereof an effective amount of the pharmaceutical composition asdescribed herein. In some embodiments, the condition is a cancer. Insome embodiments, the cancer is selected from the group consisting ofgastric cancer, sarcoma, lymphoma, leukemia, head and neck cancer,thymic cancer, epithelial cancer, salivary cancer, liver cancer, stomachcancer, thyroid cancer, lung cancer, ovarian cancer, breast cancer,prostate cancer, esophageal cancer, pancreatic cancer, glioma, leukemia,multiple myeloma, renal cell carcinoma, bladder cancer, cervical cancer,choriocarcinoma, colon cancer, oral cancer, skin cancer, and melanoma.In some embodiments, the subject is a previously treated adult patientwith locally advanced or metastatic melanoma, squamous cell head andneck cancer (SCHNC), ovarian carcinoma, sarcoma, or relapsed orrefractory classic Hodgkin's Lymphoma (cHL). In some embodiments, thecancer can be a platinum resistant and/or platinum refractory cancer,such as, for example, platinum resistant and/or refractory ovariancancer, platinum resistant and/or/refractory breast cancer, or platinumresistant and/or refractory lung cancer. In some embodiments, ananti-PD-1 antibody is administered at a dosage of about 0.5 mg/kg, about1.0 mg/kg, about 3.0 mg/kg, or about 10 mg/kg. In some embodiments, theanti-PD-1 antibody is administered once every 7, 14, 21, or 28 days. Insome embodiments, the anti-PD-1 antibody is administered intravenouslyor subcutaneously.

In another aspect, the invention provides a method of inhibiting tumorgrowth or progression in a subject who has a tumor, comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition as described herein.

In another aspect, the invention provides a method of inhibiting orpreventing metastasis of cancer cells in a subject, comprisingadministering to the subject in need thereof an effective amount of thepharmaceutical composition as described herein.

In another aspect, the invention provides a method of inducing tumorregression in a subject who has a PD-1 expressing tumor, comprisingadministering to the subject an effective amount of the pharmaceuticalcomposition as described herein.

In some embodiments, the antibody herein can be administeredparenterally in a subject. In some embodiments, the subject is a human.

In some embodiments, the method can further comprise administering aneffective amount of a second therapeutic agent. In some embodiments, thesecond therapeutic agent is, for example, crizotinib, palbociclib, ananti-CTLA4 antibody, an anti-4-1BB antibody, or a second PD-1 antibody.

Also provided is the use of any of the anti-PD-1 antagonist antibodiesprovided herein in the manufacture of a medicament for the treatment ofcancer or for inhibiting tumor growth or progression in a subject inneed thereof. In some embodiments, the anti-PD-1 antagonist antibodyreduces weight gain in the subject.

Also provided are anti-PD-1 antagonist antibodies for use in thetreatment of a cancer or for inhibiting tumor growth or progression in asubject in need thereof. In some embodiments, the cancer is, for examplewithout limitation, gastric cancer, sarcoma, lymphoma, Hodgkin'slymphoma, leukemia, head and neck cancer, thymic cancer, epithelialcancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer,lung cancer (including, for example, non-small-cell lung carcinoma),ovarian cancer, breast cancer, prostate cancer, esophageal cancer,pancreatic cancer, glioma, leukemia, multiple myeloma, renal cellcarcinoma, bladder cancer, cervical cancer, chonocarcinoma, coloncancer, oral cancer, skin cancer, and melanoma.

In another aspect, the present disclosure provides a method forenhancing the immunogenicity or therapeutic effect of a vaccine for thetreatment of a cancer in a mammal, particularly a human, which methodcomprises administering to the mammal receiving the vaccine an effectiveamount of anti-PD-1 antagonist antibody provided by the presentdisclosure.

In another aspect, the present disclosure provides a method for treatinga cancer in a mammal, particularly a human, which method comprisesadministering to the mammal (1) an effective amount of a vaccine capableof eliciting an immune response against cells of the cancer and (2) aneffective amount of an anti-PD-1 antagonist antibody provided by thepresent disclosure.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1A depicts a graph summarizing body weight of mice treated withanti-PD-1 antagonist antibody.

FIG. 1B depicts a graph summarizing body weight of mice treated withanti-PD-1 antagonist antibody.

FIG. 1C depicts a graph summarizing body weight of mice treated withanti-PD-1 antagonist antibody.

FIG. 1D depicts a graph summarizing body weight of mice treated withanti-PD-1 antagonist antibody.

FIG. 1E depicts a graph summarizing body weight of mice treated withanti-PD-1 antagonist antibody.

FIG. 2A depicts a graph summarizing EC50 for anti-PD-1 antibody bindingto primary human activated T cells.

FIG. 2B depicts a graph summarizing EC50 for anti-PD-1 antibody bindingto primary cyno activated T cells.

FIG. 3 depicts a bar graph summarizing proliferation of culturedactivated CD4 T cells treated as follows (a) no antibody; (b) isotypecontrol; (c) EH12.1; (d) C1; (e) C2; (f) C3; (g) mAb1; (h) mAbX; (i)mAb4; (j) mAb5; (k) mAb6; (l) mAb7; (m) mAb9; (n) mAb10; (o) mAb11; (p)mAb14; (q) mAb15; (r) mAb16.

FIG. 4 depicts a bar graph summarizing proliferation of culturedactivated CD8 T cells treated as follows (a) no antibody; (b) isotypecontrol; (c) EH12.1; (d) C1; (e) C2; (f) C3; (g) mAb1; (h) mAbX; (i)mAb4; (j) mAb5; (k) mAb6; (l) mAb7; (m) mAb9; (n) mAb10; (o) mAb11; (p)mAb14; (q) mAb15; (r) mAb16.

DETAILED DESCRIPTION

Disclosed herein are antibodies that specifically bind to PD-1. Methodsof making anti-PD-1 antibodies, compositions comprising theseantibodies, and methods of using these antibodies as a medicament areprovided. Anti-PD-1 antibodies can be used to inhibit tumor progression,and can be used in the prevention and/or treatment of cancer and/orother diseases.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (AcademicPress, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

DEFINITIONS

The following terms, unless otherwise indicated, shall be understood tohave the following meanings: the term “isolated molecule” as referringto a molecule (where the molecule is, for example, a polypeptide, apolynucleotide, or an antibody) that by virtue of its origin or sourceof derivation (1) is not associated with naturally associated componentsthat accompany it in its native state, (2) is substantially free ofother molecules from the same source, e.g., species, cell from which itis expressed, library, etc., (3) is expressed by a cell from a differentspecies, or (4) does not occur in nature. Thus, a molecule that ischemically synthesized, or expressed in a cellular system different fromthe system from which it naturally originates, will be “isolated” fromits naturally associated components. A molecule also may be renderedsubstantially free of naturally associated components by isolation,using purification techniques well known in the art. Molecule purity orhomogeneity may be assayed by a number of means well known in the art.For example, the purity of a polypeptide sample may be assayed usingpolyacrylamide gel electrophoresis and staining of the gel to visualizethe polypeptide using techniques well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso, unless otherwise specified, any antigen binding portion thereofthat competes with the intact antibody for specific binding, fusionproteins comprising an antigen binding portion, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site. Antigen binding portions include, for example, Fab,Fab′, F(ab′)₂, Fd, Fv, domain antibodies (dAbs, e.g., shark and camelidantibodies), fragments including complementarity determining regions(CDRs), single chain variable fragment antibodies (scFv), maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide. An antibody includes an antibody of any class, such asIgG, IgA, or IgM (or sub-class thereof), and the antibody need not be ofany particular class. Depending on the antibody amino acid sequence ofthe constant region of its heavy chains, immunoglobulins can be assignedto different classes. There are five major classes of immunoglobulins:IgA, IgD, IgE, IgG, and IgM, and several of these may be further dividedinto subclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ and IgA₂.The heavy-chain constant regions that correspond to the differentclasses of immunoglobulins are called alpha, delta, epsilon, gamma, andmu, respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chains each consist of four frameworkregions (FRs) connected by three complementarity determining regions(CDRs) also known as hypervariable regions, and contribute to theformation of the antigen binding site of antibodies. If variants of asubject variable region are desired, particularly with substitution inamino acid residues outside of a CDR region (i.e., in the frameworkregion), appropriate amino acid substitution, preferably, conservativeamino acid substitution, can be identified by comparing the subjectvariable region to the variable regions of other antibodies whichcontain CDR1 and CDR2 sequences in the same canonincal class as thesubject variable region (Chothia and Lesk, J Mol Biol 196(4): 901-917,1987).

In certain embodiments, definitive delineation of a CDR andidentification of residues comprising the binding site of an antibody isaccomplished by solving the structure of the antibody and/or solving thestructure of the antibody-ligand complex. In certain embodiments, thatcan be accomplished by any of a variety of techniques known to thoseskilled in the art, such as X-ray crystallography. In certainembodiments, various methods of analysis can be employed to identify orapproximate the CDR regions. In certain embodiments, various methods ofanalysis can be employed to identify or approximate the CDR regions.Examples of such methods include, but are not limited to, the Kabatdefinition, the Chothia definition, the AbM definition, the contactdefinition, and the conformational definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See, e.g.,Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17;Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See, e.g., Martin et al., 1989, Proc NatiAcad Sci (USA), 86:9268-9272; “AbM™, A Computer Program for ModelingVariable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. TheAbM definition models the tertiary structure of an antibody from primarysequence using a combination of knowledge databases and ab initiomethods, such as those described by Samudrala et al., 1999, “Ab InitioProtein Structure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198. Thecontact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol.,5:732-45. In another approach, referred to herein as the “conformationaldefinition” of CDRs, the positions of the CDRs may be identified as theresidues that make enthalpic contributions to antigen binding. See,e.g., Makabe et al., 2008, Journal of Biological Chemistry,283:1156-1166. Still other CDR boundary definitions may not strictlyfollow one of the above approaches, but will nonetheless overlap with atleast a portion of the Kabat CDRs, although they may be shortened orlengthened in light of prediction or experimental findings thatparticular residues or groups of residues do not significantly impactantigen binding. As used herein, a CDR may refer to CDRs defined by anyapproach known in the art, including combinations of approaches. Themethods used herein may utilize CDRs defined according to any of theseapproaches. For any given embodiment containing more than one CDR, theCDRs may be defined in accordance with any of Kabat, Chothia, extended,AbM, contact, and/or conformational definitions.

As known in the art, a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature 348:552-554, for example. As used herein, “humanized”antibody refers to forms of non-human (e.g. murine) antibodies that arechimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) that contain minimal sequence derived from non-humanimmunoglobulin. Preferably, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from a CDR of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. The humanized antibody may compriseresidues that are found neither in the recipient antibody nor in theimported CDR or framework sequences, but are included to further refineand optimize antibody performance.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen bindingresidues.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The term “epitope” refers to that portion of a molecule capable of beingrecognized by and bound by an antibody at one or more of the antibody'santigen-binding regions. Epitopes often consist of a surface grouping ofmolecules such as amino acids or sugar side chains and have specificthree-dimensional structural characteristics as well as specific chargecharacteristics. In some embodiments, the epitope can be a proteinepitope. Protein epitopes can be linear or conformational. In a linearepitope, all of the points of interaction between the protein and theinteracting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. A “nonlinear epitope” or“conformational epitope” comprises noncontiguous polypeptides (or aminoacids) within the antigenic protein to which an antibody specific to theepitope binds. The term “antigenic epitope” as used herein, is definedas a portion of an antigen to which an antibody can specifically bind asdetermined by any method well known in the art, for example, byconventional immunoassays. Once a desired epitope on an antigen isdetermined, it is possible to generate antibodies to that epitope, e.g.,using the techniques described in the present specification.Alternatively, during the discovery process, the generation andcharacterization of antibodies may elucidate information about desirableepitopes. From this information, it is then possible to competitivelyscreen antibodies for binding to the same epitope. An approach toachieve this is to conduct competition and cross-competition studies tofind antibodies that compete or cross-compete with one another forbinding to PD-1, e.g., the antibodies compete for binding to theantigen.

As used herein, the term “PD-1” refers to any form of PD-1 and variantsthereof that retain at least part of the activity of PD-1. Unlessindicated differently, such as by specific reference to human PD-1, PD-1includes all mammalian species of native sequence PD-1, e.g., human,canine, feline, equine, and bovine. One exemplary human PD-1 is found asUniprot Accession Number Q15116 (SEQ ID NO: 1).

The term “agonist” refers to a substance which promotes (i.e., induces,causes, enhances, or increases) the biological activity or effect ofanother molecule. The term agonist encompasses substances which bindreceptor, such as an antibody, and substances which promote receptorfunction without binding thereto (e.g., by activating an associatedprotein).

The term “antagonist” or “inhibitor” refers to a substance thatprevents, blocks, inhibits, neutralizes, or reduces a biologicalactivity or effect of another molecule, such as a receptor.

The term “antagonist antibody” refers to an antibody that binds to atarget and prevents or reduces the biological effect of that target. Insome embodiments, the term can denote an antibody that prevents thetarget, e.g., PD-1, to which it is bound from performing a biologicalfunction.

As used herein, an “anti-PD-1 antagonist antibody” refers to an antibodythat is able to inhibit PD-1 biological activity and/or downstreamevents(s) mediated by PD-1. Anti-PD-1 antagonist antibodies encompassantibodies that block, antagonize, suppress or reduce (to any degreeincluding significantly) PD-1 biological activity, including downstreamevents mediated by PD-1, such PD-L1 binding and downstream signaling,PD-L2 binding and downstream signaling, inhibition of T cellproliferation, inhibition of T cell activation, inhibition of IFNsecretion, inhibition of IL-2 secretion, inhibition of TNF secretion,induction of IL-10, and inhibition of anti-tumor immune responses. Forpurposes of the present invention, it will be explicitly understood thatthe term “anti-PD-1 antagonist antibody” (interchangeably termed“antagonist PD-1 antibody”, “antagonist anti-PD-1 antibody” or “PD-1antagonist antibody”) encompasses all the previously identified terms,titles, and functional states and characteristics whereby PD-1 itself, aPD-1 biological activity, or the consequences of the biologicalactivity, are substantially nullified, decreased, or neutralized in anymeaningful degree. In some embodiments, an anti-PD-1 antagonist antibodybinds PD-1 and upregulates an anti-tumor immune response. Examples ofanti-PD-1 antagonist antibodies are provided herein.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. The chain may be linear or branched, it may comprise modifiedamino acids, and/or may be interrupted by non-amino acids. The termsalso encompass an amino acid chain that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that the polypeptides can occur as single chains orassociated chains.

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

As used herein, an antibody “interacts with” PD-1 when the equilibriumdissociation constant is equal to or less than 20 nM, preferably lessthan about 6 nM, more preferably less than about 1 nM, most preferablyless than about 0.2 nM, as measured by the methods disclosed herein inExample 7.

An antibody that “preferentially binds” or “specifically binds” (usedinterchangeably herein) to an epitope is a term well understood in theart, and methods to determine such specific or preferential binding arealso well known in the art. A molecule is said to exhibit “specificbinding” or “preferential binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to a PD-1 epitope is an antibody that binds thisepitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other PD-1 epitopes or non-PD-1epitopes. It is also understood by reading this definition that, forexample, an antibody (or moiety or epitope) that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. Generally, but not necessarily, reference tobinding means preferential binding.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant domains, CH2 and CH3. Asis known in the art, an Fc region can be present in dimer or monomericform.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods,4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR”also includes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al., 1976, J. Immunol.,117:587; and Kim et al., 1994, J. Immunol., 24:249).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding portion thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding portion thereof, such that the result ofbinding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity; phagocytosis;down-regulation of cell surface receptors (e.g. B cell receptor), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g. an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification, yet retains at least one effector function of the nativesequence Fc region. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g. from about one to about tenamino acid substitutions, and preferably, from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably, at least about 90% sequence identity therewith, morepreferably, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99% sequence identity therewith.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: reducing the proliferation of (or destroying) neoplasticor cancerous cells, inhibiting metastasis of neoplastic cells, shrinkingor decreasing the size of a tumor, remission of cancer, decreasingsymptoms resulting from cancer, increasing the quality of life of thosesuffering from cancer, decreasing the dose of other medications requiredto treat cancer, delaying the progression of cancer, curing a cancer,and/or prolong survival of patients having cancer.

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an anti-PD-1 antagonist antibody.“Ameliorating” also includes shortening or reduction in duration of asymptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. In more specificaspects, an effective amount prevents, alleviates or amelioratessymptoms of disease, and/or prolongs the survival of the subject beingtreated. For prophylactic use, beneficial or desired results includeeliminating or reducing the risk, lessening the severity, or delayingthe outset of the disease, including biochemical, histological and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease.For therapeutic use, beneficial or desired results include clinicalresults such as reducing one or more symptoms of a disease such as, forexample, cancer including, for example without limitation, gastriccancer, sarcoma, lymphoma, Hodgkin's lymphoma, leukemia, head and neckcancer, squamous cell head and neck cancer, thymic cancer, epithelialcancer, salivary cancer, liver cancer, stomach cancer, thyroid cancer,lung cancer, ovarian cancer, breast cancer, prostate cancer, esophagealcancer, pancreatic cancer, glioma, leukemia, multiple myeloma, renalcell carcinoma, bladder cancer, cervical cancer, choriocarcinoma, coloncancer, oral cancer, skin cancer, and melanoma, decreasing the dose ofother medications required to treat the disease, enhancing the effect ofanother medication, and/or delaying the progression of the cancer inpatients. An effective dosage can be administered in one or moreadministrations. For purposes of this invention, an effective dosage ofdrug, compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. As is understood in the clinical context, an effectivedosage of a drug, compound, or pharmaceutical composition may or may notbe achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to, farm animals (e.g., cows,pigs, horses, chickens, etc.), sport animals, pets, primates, horses,dogs, cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

The term “k_(on)”, as used herein, refers to the rate constant forassociation of an antibody to an antigen. Specifically, the rateconstants (k_(on) and k_(off)) and equilibrium dissociation constantsare measured using full-length antibodies and/or Fab antibody fragments(i.e. univalent) and PD-1.

The term “k_(off)”, as used herein, refers to the rate constant fordissociation of an antibody from the antibody/antigen complex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.

The term “immune-effector-cell enhancer” or “IEC enhancer” refers to asubstance capable of increasing or enhancing the number, quality, orfunction of one or more types of immune effector cells of a mammal.Examples of immune effector cells include cytolytic CD8 T cells, CD4 Tcells, NK cells, and B cells.

The term “immune modulator” refers to a substance capable of altering(e.g., inhibiting, decreasing, increasing, enhancing, or stimulating)the immune response (as defined herein) or the working of any componentof the innate, humoral or cellular immune system of a host mammal. Thus,the term “immune modulator” encompasses the “immune-effector-cellenhancer” as defined herein and the “immune-suppressive-cell inhibitor”as defined herein, as well as substance that affects other components ofthe immune system of a mammal.

The term “immune response” refers to any detectable response to aparticular substance (such as an antigen or immunogen) by the immunesystem of a host mammal, such as innate immune responses (e.g.,activation of Toll receptor signaling cascade), cell-mediated immuneresponses (e.g., responses mediated by T cells, such as antigen-specificT cells, and non-specific cells of the immune system), and humoralimmune responses (e.g., responses mediated by B cells, such asgeneration and secretion of antibodies into the plasma, lymph, and/ortissue fluids).

The term “immunogenic” refers to the ability of a substance to cause,elicit, stimulate, or induce an immune response, or to improve, enhance,increase or prolong a pre-existing immune response, against a particularantigen, whether alone or when linked to a carrier, in the presence orabsence of an adjuvant.

The term “immune-suppressive-cell inhibitor” or “ISC inhibitor” refersto a substance capable of reducing or suppressing the number or functionof immune suppressive cells of a mammal. Examples of immune suppressivecells include regulatory T cells (“T regs”), myeloid-derived suppressorcells, and tumor-associated macrophages.

The term “intradermal administration,” or “administered intradermally,”in the context of administering a substance to a mammal including ahuman, refers to the delivery of the substance into the dermis layer ofthe skin of the mammal. The skin of a mammal is composed of an epidermislayer, a dermis layer, and a subcutaneous layer. The epidermis is theouter layer of the skin. The dermis, which is the middle layer of theskin, contains nerve endings, sweat glands and oil (sebaceous) glands,hair follicles, and blood vessels. The subcutaneous layer is made up offat and connective tissue that houses larger blood vessels and nerves.In contrast in intradermal administration, “subcutaneous administration”refers to the administration of a substance into the subcutaneous layerand “topical administration” refers to the administration of a substanceonto the surface of the skin.

The term “neoplastic disorder” refers to a condition in which cellsproliferate at an abnormally high and uncontrolled rate, the rateexceeding and uncoordinated with that of the surrounding normal tissues.It usually results in a solid lesion or lump known as “tumor.” This termencompasses benign and malignant neoplastic disorders. The term“malignant neoplastic disorder”, which is used interchangeably with theterm “cancer” in the present disclosure, refers to a neoplastic disordercharacterized by the ability of the tumor cells to spread to otherlocations in the body (known as “metastasis”). The term “benignneoplastic disorder” refers to a neoplastic disorder in which the tumorcells lack the ability to metastasize.

The term “preventing” or “prevent” refers to (a) keeping a disorder fromoccurring or (b) delaying the onset of a disorder or onset of symptomsof a disorder.

The term “tumor-associated antigen” or “TAA” refers to an antigen whichis specifically expressed by tumor cells or expressed at a higherfrequency or density by tumor cells than by non-tumor cells of the sametissue type. Tumor-associated antigens may be antigens not normallyexpressed by the host; they may be mutated, truncated, misfolded, orotherwise abnormal manifestations of molecules normally expressed by thehost; they may be identical to molecules normally expressed butexpressed at abnormally high levels; or they may be expressed in acontext or milieu that is abnormal. Tumor-associated antigens may be,for example, proteins or protein fragments, complex carbohydrates,gangliosides, haptens, nucleic acids, or any combination of these orother biological molecules.

The term “vaccine” refers to an immunogenic composition foradministration to a mammal for eliciting an immune response against aparticular antigen in the mammal. A vaccine typically contains an agent(known as “antigen” or “immunogen”) that resembles, or is derived from,the target of the immune response, such as a disease-causingmicro-organism or tumor cells. A vaccine intended for the treatment of atumor, such as a cancer, typically contains an antigen that is derivedfrom a TAA found on the target tumor and is able to elicitimmunogenicity against the TAA on the target tumor.

The term “vaccine-based immunotherapy regimen” refers to a therapeuticregimen in which a vaccine is administered in combination with one ormore immune modulators. The vaccine and the immune modulators may beadministered together in a single formulation or administered separately

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular. Any example(s) following the term “e.g.” or “forexample” is not meant to be exhaustive or limiting.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Anti-PD-1 Antagonist Antibodies

Provided herein are anti-PD-1 antagonist antibodies that block, suppressor reduce (including significantly reduces) PD-1 biological activity,including downstream events mediated by PD-1. An anti-PD-1 antagonistantibody should exhibit any one or more of the followingcharacteristics: (a) bind to PD-1 and block downstream signaling events;(b) block PD-L1 binding to PD-1; (c) upregulate a T cell-mediated immuneresponse; (d) stimulate IFNγ secretion; (e) stimulate TNF secretion; (f)increase T cell proliferation; and (g) reduce inhibitory signaltransduction through PD-1.

For purposes of this invention, the antibody preferably reacts with PD-1in a manner that inhibits PD-1 signaling function. In some embodiments,the anti-PD-1 antagonist antibody specifically binds primate PD-1.

The antibodies useful in the present invention can encompass monoclonalantibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab′,F(ab′)₂, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies,heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusionproteins comprising an antibody portion (e.g., a domain antibody),humanized antibodies, and any other modified configuration of theimmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity, including glycosylation variants ofantibodies, amino acid sequence variants of antibodies, and covalentlymodified antibodies. The antibodies may be murine, rat, human, or anyother origin (including chimeric or humanized antibodies). In someembodiments, the anti-PD-1 antagonist antibody is a monoclonal antibody.In some embodiments, the antibody is a human or humanized antibody.

The anti-PD-1 antagonist antibodies may be made by any method known inthe art. General techniques for production of human and mouse antibodiesare known in the art and/or are described herein.

Anti-PD-1 antagonist antibodies can be identified or characterized usingmethods known in the art, whereby reduction, amelioration, orneutralization of PD-1 biological activity is detected and/or measured.In some embodiments, an anti-PD-1 antagonist antibody is identified byincubating a candidate agent with PD-1 and monitoring binding and/orattendant reduction or neutralization of a biological activity of PD-1.The binding assay may be performed with, e.g., purified PD-1polypeptide(s), or with cells naturally expressing (e.g., variousstrains), or transfected to express, PD-1 polypeptide(s). In oneembodiment, the binding assay is a competitive binding assay, where theability of a candidate antibody to compete with a known anti-PD-1antagonist antibody for PD-1 binding is evaluated. The assay may beperformed in various formats, including the ELISA format. In someembodiments, an anti-PD-1 antagonist antibody is identified byincubating a candidate antibody with PD-1 and monitoring binding.

Following initial identification, the activity of a candidate anti-PD-1antagonist antibody can be further confirmed and refined by bioassays,known to test the targeted biological activities. In some embodiments,an in vitro cell assay is used to further characterize a candidateanti-PD-1 antagonist antibody. For example, a candidate antibody isincubated with primary human T cells, and PD-L1 is added, and IFNγsecretion is monitored. Alternatively, bioassays can be used to screencandidates directly.

The anti-PD-1 antagonist antibodies of the invention exhibit one or moreof the following characteristics: (a) bind to PD-1 and block downstreamsignaling events; (b) block PD-L1 binding to PD-1; (c) upregulate a Tcell-mediated immune response; (d) stimulate IFNγ secretion; (e)stimulate TNF secretion; (f) increase T cell proliferation; (g) reduceinhibitory signal transduction through PD-1; and (h) block PD-L2 bindingto PD-1. Preferably, anti-PD-1 antibodies have two or more of thesefeatures. More preferably, the antibodies have three or more of thefeatures. More preferably, the antibodies have four or more of thefeatures. More preferably, the antibodies have five or more of thefeatures. More preferably, the antibodies have six or more of thefeatures. More preferably, the antibodies have seven or more of thefeatures. Most preferably, the antibodies have all eightcharacteristics.

Anti-PD-1 antagonist antibodies may be characterized using methods wellknown in the art. For example, one method is to identify the epitope towhich it binds, or “epitope mapping.” There are many methods known inthe art for mapping and characterizing the location of epitopes onproteins, including solving the crystal structure of an antibody-antigencomplex, competition assays, gene fragment expression assays, andsynthetic peptide-based assays, as described, for example, in Chapter 11of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N. Y., 1999. In anadditional example, epitope mapping can be used to determine thesequence to which an anti-PD-1 antagonist antibody binds. Epitopemapping is commercially available from various sources, for example,Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). Theepitope can be a linear epitope, i.e., contained in a single stretch ofamino acids, or a conformational epitope formed by a three-dimensionalinteraction of amino acids that may not necessarily be contained in asingle stretch. Peptides of varying lengths (e.g., at least 4-6 aminoacids long) can be isolated or synthesized (e.g., recombinantly) andused for binding assays with an anti-PD-1 antagonist antibody. Inanother example, the epitope to which the anti-PD-1 antagonist antibodybinds can be determined in a systematic screening by using overlappingpeptides derived from the PD-1 sequence and determining binding by theanti-PD-1 antagonist antibody. According to the gene fragment expressionassays, the open reading frame encoding PD-1 is fragmented eitherrandomly or by specific genetic constructions and the reactivity of theexpressed fragments of PD-1 with the antibody to be tested isdetermined. The gene fragments may, for example, be produced by PCR andthen transcribed and translated into protein in vitro, in the presenceof radioactive amino acids. The binding of the antibody to theradioactively labeled PD-1 fragments is then determined byimmunoprecipitation and gel electrophoresis. Certain epitopes can alsobe identified by using large libraries of random peptide sequencesdisplayed on the surface of phage particles (phage libraries) or yeast(yeast display). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays. In an additional example, mutagenesis of an antigen,domain swapping experiments and alanine scanning mutagenesis can beperformed to identify residues required, sufficient, and/or necessaryfor epitope binding. For example, alanine scanning mutagenesisexperiments can be performed using a mutant PD-1 in which variousresidues of the PD-1 polypeptide have been replaced with alanine. Byassessing binding of the antibody to the mutant PD-1, the importance ofthe particular PD-1 residues to antibody binding can be assessed.

Yet another method which can be used to characterize an anti-PD-1antagonist antibody is to use competition assays with other antibodiesknown to bind to the same antigen, i.e., various fragments of PD-1, todetermine if the anti-PD-1 antagonist antibody binds to the same epitopeas other antibodies. Competition assays are well known to those of skillin the art, including in an ELISA format.

The binding affinity (K_(D)) of an anti-PD-1 antagonist antibody to PD-1can be about 0.001 to about 200 nM. In some embodiments, the bindingaffinity is any of about 200 nM, about 100 nM, about 50 nM, about 10 nM,about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about20 pM, about 15 pM, about 10 pM, about 5 pM, about 2 pM, or about 1 pM.In some embodiments, the binding affinity is less than any of about 250nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM,about 500 pM, about 100 pM, about 50 pM, about 20 pM, about 10 pM, about5 pM, or about 2 pM.

Accordingly, the invention provides any of the following, orcompositions (including pharmaceutical compositions) comprising anantibody having a partial light chain sequence and a partial heavy chainsequence as found in Table 1, or variants thereof. In Table 1, theunderlined sequences are CDR sequences. In Table 1, the K_(D) indicatesaffinity for human PD-1 as measured using surface plasmon resonance at25° C., unless indicated otherwise.

TABLE 1 Variable Regions Sequences of Anti-PD-1 antagonist Antibodies KDmAb Light Chain Heavy Chain (nM) mAb1 DIVMTQSPDSLAVSLGERAQVQLVQSGAEVKKPGASVKVS 64.24 TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAPLTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSLTNYN STRESGVPDRFSGSGSGTEKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLLTGTFNDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID (SEQ ID NO: 2) NO: 3) mAb2DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 2.22 TINCKSSQSLWDSGNQKNFCKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSLTNYNSYRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQLSSLRSEDTAVYYCARLLTGTF NDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID(SEQ ID NO: 7) NO: 3) mAb3 DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS1.43 TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWTGQGLEWMGNIYPGSSLTNYN SYRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYMEDFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLLTGTF NDYFYPHTFGGGTKVEIKAYWGQGTLVTVSS (SEQ ID (SEQ ID NO: 8) NO: 3) mAb4 DIVMTQSPDSLAVSLGERAQVQLVQSGAEVKKPGASVKVS 89 (at TINCKSSQSLWDSTNQKNF CKASGYTFTSYWINWVRQAP37° C.) LTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSLTNYN STRESGVPDRFSGSGSGTEKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLLTGTFNDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID (SEQ ID NO: 9) NO: 3) mAb5DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 12.82 TINCKSSQSLWDSGNQKNFCKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSLTNYNSTRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQLSSLRSEDTAVYYCARLSTGTF NDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID NO: 2)(SEQ ID NO: 4) mAb6 DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 1.16TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWTGQGLEWMGNIYPGSSLTNYN SYRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYMEDFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLSTGTF NDYFYPLTFGGGTKVEIKAYWGQGTLVTVSS (SEQ ID NO: 7) (SEQ ID NO: 4) mAb7 DIVMTQSPDSLAVSLGERAQVQLVQSGAEVKKPGASVKVS 0.73 TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAPLTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSLTNYN SYRESGVPDRFSGSGSGTEKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLSTGTFNDYFYPHTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID NO: 8) (SEQ ID NO: 4) mAb8DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 17.35 TINCKSSQSLWDSTNQKNFCKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSLTNYNSTRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQLSSLRSEDTAVYYCARLSTGTF NDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID NO: 9)(SEQ ID NO: 4) mAb9 DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 13.54TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWTGQGLEWMGNIYPGSSITNYNE STRESGVPDRFSGSGSGT KFKNRVTMTRDTSTSTVYMELDFTLTISSLQAEDVAVYYCQ SSLRSEDTAVYYCARLTTGTF NDYFYPLTFGGGTKVEIKAYWGQGTLVTVSS (SEQ ID (SEQ ID NO: 2) NO: 5) mAb10 DIVMTQSPDSLAVSLGERAQVQLVQSGAEVKKPGASVKVS 0.98 TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAPLTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSITNYNE SYRESGVPDRFSGSGSGTKFKNRVTMTRDTSTSTVYMEL DFTLTISSLQAEDVAVYYCQ SSLRSEDTAVYYCARLTTGTFNDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID (SEQ ID NO: 7) NO: 5) mAb11DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 0.93 TINCKSSQSLWDSGNQKNFCKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWT GQGLEWMGNIYPGSSITNYNESYRESGVPDRFSGSGSGT KFKNRVTMTRDTSTSTVYMEL DFTLTISSLQAEDVAVYYCQSSLRSEDTAVYYCARLTTGTF NDYFYPHTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID(SEQ ID NO: 8) NO: 5) mAb12 DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS17.27 TINCKSSQSLWDSTNQKNF CKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWTGQGLEWMGNIYPGSSITNYNE STRESGVPDRFSGSGSGT KFKNRVTMTRDTSTSTVYMELDFTLTISSLQAEDVAVYYCQ SSLRSEDTAVYYCARLTTGTF NDYFYPLTFGGGTKVEIKAYWGQGTLVTVSS (SEQ ID (SEQ ID NO: 9) NO: 5) mAb13 DIVMTQSPDSLAVSLGERAQVQLVQSGAEVKKPGASVKVS 5.87 TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAPLTWYQQKPGQPPKLLIYWT GQGLEWMGNIWPGSSLTNYN STRESGVPDRFSGSGSGTEKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLLTGTFNDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID NO: 2) (SEQ ID NO: 6) mAb14DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 0.6 TINCKSSQSLWDSGNQKNFCKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWT GQGLEWMGNIWPGSSLTNYNSYRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQLSSLRSEDTAVYYCARLLTGTF NDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID NO: 7)(SEQ ID NO: 6) mAb15 DIVMTQSPDSLAVSLGERA QVQLVQSGAEVKKPGASVKVS 0.49TINCKSSQSLWDSGNQKNF CKASGYTFTSYWINWVRQAP LTWYQQKPGQPPKLLIYWTGQGLEWMGNIWPGSSLTNYN SYRESGVPDRFSGSGSGT EKFKNRVTMTRDTSTSTVYMEDFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLLTGTF NDYFYPHTFGGGTKVEIKAYWGQGTLVTVSS (SEQ ID NO: 8) (SEQ ID NO: 6) mAb16 DIVMTQSPDSLAVSLGERAQVQLVQSGAEVKKPGASVKVS 7.51 TINCKSSQSLWDSTNQKNF CKASGYTFTSYWINWVRQAPLTWYQQKPGQPPKLLIYWT GQGLEWMGNIWPGSSLTNYN STRESGVPDRFSGSGSGTEKFKNRVTMTRDTSTSTVYME DFTLTISSLQAEDVAVYYCQ LSSLRSEDTAVYYCARLLTGTFNDYFYPLTFGGGTKVEIK AYWGQGTLVTVSS (SEQ ID NO: 9) (SEQ ID NO: 6)

The invention also provides CDR portions of antibodies to PD-1.Determination of CDR regions is well within the skill of the art. It isunderstood that in some embodiments, CDRs can be a combination of theKabat and Chothia CDR (also termed “combined CDRs” or “extended CDRs”).In another approach, referred to herein as the “conformationaldefinition” of CDRs, the positions of the CDRs may be identified as theresidues that make enthalpic contributions to antigen binding. See,e.g., Makabe et al., 2008, Journal of Biological Chemistry,283:1156-1166. In general, “conformational CDRs” include the residuepositions in the Kabat CDRs and Vernier zones which are constrained inorder to maintain proper loop structure for the antibody to bind aspecific antigen. Determination of conformational CDRs is well withinthe skill of the art. In some embodiments, the CDRs are the Kabat CDRs.In other embodiments, the CDRs are the Chothia CDRs. In otherembodiments, the CDRs are the extended, AbM, conformational, or contactCDRs. In other words, in embodiments with more than one CDR, the CDRsmay be any of Kabat, Chothia, extended, AbM, conformational, contactCDRs or combinations thereof.

In some embodiments, the antibody comprises three CDRs of any one of theheavy chain variable regions shown in Table 1. In some embodiments, theantibody comprises three CDRs of any one of the light chain variableregions shown in Table 1. In some embodiments, the antibody comprisesthree CDRs of any one of the heavy chain variable regions shown in Table1, and three CDRs of any one of the light chain variable regions shownin Table 1.

Table 2 provides examples of CDR sequences of anti-PD-1 antagonistantibodies provided herein.

TABLE 2 Anti-PD-1 antagonist antibodies (mAbs) and theirantigen-binding CDR sequences according toKabat (underlined) and Chothia (bold) mAb CDR1 CDR2 CDR3 mAb1 LKSSQSLWDSGNQKN WTSTRES QNDYFYPLT FLT  (SEQ ID NO: 10) (SEQ ID NO: 11)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ NIYPGSSLTNYNEKFK LLTGTFAYID NOs: 13 (whole), 14 N (SEQ ID NOs: 16 and (SEQ ID NO: 18) and 15) 17)mAb2 L KSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPLT FLT (SEQ ID NO: 10) 20) (SEQ ID NO: 12) H GYTFT SYWIN (SEQ NIYPGSSLTNYNEKFKLLTGTFAY ID NOs: 13 (whole), 14 N (SEQ ID NOs: 16 and (SEQ ID NO: 18)and 15) 17) mAb3 L KSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPHT FLT (SEQ ID NO: 10) 20) (SEQ ID NO: 21) H GYTFT SYWIN (SEQ NIYPGSSLTNYNEKFKLLTGTFAY ID NOs: 13 (whole), 14 N (SEQ ID NOs: 16 and (SEQ ID NO: 18)and 15) 17) mAb4 L KSSQSLWDSTNQKNF WTSTRES  (SEQ ID NO: QNDYFYPLT LT (SEQ ID NO: 22) 11) (SEQ ID NO: 12) H GYTFT SYWIN (SEQ NIYPGSSLTNYNEKFKLLTGTFAY ID NOs: 13 (whole), 14 N (SEQ ID NOs: 16 and (SEQ ID NO: 18)and 15) 17) mAb5 L KSSQSLWDSGNQKN WTSTRES  (SEQ ID NO: QNDYFYPLT FLT (SEQ ID NO: 10) 11) (SEQ ID NO: 12) H GYTFT SYWIN (SEQ IDNIYPGSSLTNYNEKFK LSTGTFAY  (SEQ NOs: 13 (whole), 14N (SEQ ID NOs: 16 and ID NO: 23) and 15) 17) mAb6 L KSSQSLWDSGNQKNWTSYRES  (SEQ ID NO: QNDYFYPLT FLT  (SEQ ID NO: 10) 20) (SEQ ID NO: 12)H GYTFT SYWIN (SEQ ID NIYPGSSLTNYNEKFK LSTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NOs: 16 and ID NO: 23) and 15) 17) mAb7 LKSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPHT FLT  (SEQ ID NO: 10) 20)(SEQ ID NO: 21) H GYTFT SYWIN (SEQ ID NIYPGSSLTNYNEKFK LSTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NOs: 16 and ID NO: 23) and 15) 17) mAb8 LKSSQSLWDSTNQKNF WTSTRES  (SEQ ID NO: QNDYFYPLT LT  (SEQ ID NO: 22) 11)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIYPGSSLTNYNEKFK LSTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NOs: 16 and ID NO: 23) and 15) 17) mAb9 LKSSQSLWDSGNQKN WTSTRES (SEQ ID NO: QNDYFYPLT FLT  (SEQ ID NO: 10) 11)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIYPGSSITNYNEKFKN LTTGTFAY  (SEQNOs: 13 (whole), 14 (SEQ ID NO: 24 and 25) ID NO: 26) and 15) mAb10 LKSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPLT FLT  (SEQ ID NO: 10) 20)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIYPGSSITNYNEKFKN LTTGTFAY  (SEQNOs: 13 (whole), 14 (SEQ ID NO: 24 and 25) ID NO: 26) and 15) mAb11 LKSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPHT FLT  (SEQ ID NO: 10) 20)(SEQ ID NO: 21) H GYTFT SYWIN (SEQ ID NIYPGSSITNYNEKFKN LTTGTFAY  (SEQNOs: 13 (whole), 14 (SEQ ID NO: 24 and 25) ID NO: 26) and 15) mAb12 LKSSQSLWDSTNQKNF WTSTRES  (SEQ ID NO: QNDYFYPLT LT  (SEQ ID NO: 22) 11)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIYPGSSITNYNEKFKN LTTGTFAY  (SEQNOs: 13 (whole), 14 (SEQ ID NO: 24 and 25) ID NO: 26) and 15) mAb13 LKSSQSLWDSGNQKN WTSTRES  (SEQ ID NO: QNDYFYPLT FLT  (SEQ ID NO: 10) 11)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIWPGSSLTNYNEKFK LLTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NO: 27 and ID NO: 18) and 15) 28) mAb14 LKSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPLT FLT  (SEQ ID NO: 10) 20)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIWPGSSLTNYNEKFK LLTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NO: 27 and ID NO: 18) and 15) 28) mAb15 LKSSQSLWDSGNQKN WTSYRES  (SEQ ID NO: QNDYFYPHT FLT  (SEQ ID NO: 10) 20)(SEQ ID NO: 21) H GYTFT SYWIN (SEQ ID NIWPGSSLTNYNEKFK LLTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NO: 27 and ID NO: 18) and 15) 28) mAb16 LKSSQSLWDSTNQKNF WTSTRES  (SEQ ID NO: QNDYFYPLT LT  (SEQ ID NO: 22) 11)(SEQ ID NO: 12) H GYTFT SYWIN (SEQ ID NIWPGSSLTNYNEKFK LLTGTFAY  (SEQNOs: 13 (whole), 14 N (SEQ ID NO: 27 and ID NO: 18) and 15) 28)In some embodiments, the antibody comprises three light chain CDRs andthree heavy chain CDRs from Table 2.

An alignment of light chain CDRs from anti-PD-1 antibodies is providedin Table 3. Variable residues are shown in bold. Consensus light chainCDR sequences are provided in the last row of Table 3.

TABLE 3 Alignment of anti-PD-1 light chain CDRs SEQ SEQ SEQ ID ID mAbVL CDR1 ID NO: VL CDR2 NO: VL CDR3 NO: 1, 5, KSSQSLWDSGNQK 10 WTSTRES 11QNDYFYPLT 12 9, 13 NFLT 2, 6, KSSQSLWDSGNQK 10 WTSYRES 20 QNDYFYPLT 1210, 14 NFLT 3, 7, KSSQSLWDSGNQK 10 WTSYRES 20 QNDYFYPHT 21 11, 15 NFLT4, 8, KSSQSLWDSTNQK 22 WTSTRES 11 QNDYFYPLT 12 12, 16 NFLT 17KSSQSLLDSGNQK 30 WTSTRES 11 QNDYSYPLT 31 NFLT KSSQSLX ₁DSX ₂NQ 32 WTSX₁RE 33 QNDY X ₁YP 34 KNFLT, wherein X ₁ is S, wherein X ₂T, wherein X ₁W or L, and X ₂ is G X ₁ is T or Y is F or S, and X ₂ or T is L or H

An alignment of heavy chain CDRs from anti-PD-1 antibodies is providedin Table 4. Variable residues are shown in bold. Consensus heavy chainCDR sequences are provided in the last row of Table 4.

TABLE 4 Alignment of anti-PD-1 heavy chain CDRs SEQ SEQ SEQ ID ID ID mAbVH CDR1 NO: VH CDR2 NO: VH CDR3 NO: 1-4 GYTFTSYWIN 13 NIYPGSSLTNYNEKFKN17 LLTGTFAY 18 5-8 GYTFTSYWIN 13 NIYPGSSLTNYNEKFKN 17 LSTGTFAY 23  9-12GYTFTSYWIN 13 NIYPGSSITNYNEKFKN 25 LTTGTFAY 26 13-16 GYTFTSYWIN 13NIWPGSSLTNYNEKFKN 28 LLTGTFAY 18 17 GYTFTSYWIN 13 NIYPGSSSTNYNEKFKN 35LLTGTFAY 18 GYTFTSYWIN 13 NIX ₁PGSSX ₂TNYNEKFKN, 36 LX ₁TGTFAY, 37wherein X ₁ is Y or W, and wherein X ₁ X ₂ is L, I, or S is L or S

In some embodiments, the antibody comprises three light chain CDRs fromTable 3 and three heavy chain CDRs from Table 4.

In some embodiments, the antibody comprises the full-length heavy chain,with or without the C-terminal lysine, and/or the full-length lightchain of anti-PD-1 antagonist antibody mAb7 or mAb15. The amino acidsequence of mAb7 full-length heavy chain (SEQ ID NO: 29) is shown below:

(SEQ ID NO: 29) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK

The amino acid sequence of mAb7 full-length heavy chain without theC-terminal lysine (SEQ ID NO: 38) is shown below:

(SEQ ID NO: 38) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWMGNIYPGSSLTNYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSTGTFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY TQKSLSLSLG

The amino acid sequence of mAb7 full-length light chain (SEQ ID NO: 39)is shown below:

(SEQ ID NO: 39) DIVMTQSPDSLAVSLGERATINCKSSQSLWDSGNQKNFLTWYQQKPGQPPKLLIYWTSYRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPHTFGGGTKVEIKRGTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The invention also provides methods of generating, selecting, and makinganti-PD-1 antagonist antibodies. The antibodies of this invention can bemade by procedures known in the art. In some embodiments, antibodies maybe made recombinantly and expressed using any method known in the art.

In some embodiments, antibodies may be prepared and selected by phagedisplay technology. See, for example, U.S. Pat. Nos. 5,565,332;5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev.Immunol. 12:433-455, 1994. Alternatively, the phage display technology(McCafferty et al., Nature 348:552-553, 1990) can be used to producehuman antibodies and antibody fragments in vitro, from immunoglobulinvariable (V) domain gene repertoires from unimmunized donors. Accordingto this technique, antibody V domain genes are cloned in-frame intoeither a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B cell.Phage display can be performed in a variety of formats; for review see,e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3:564-571, 1993. Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature 352:624-628,1991, isolated a diverse array of anti-oxazolone antibodies from a smallrandom combinatorial library of V genes derived from the spleens ofimmunized mice. A repertoire of V genes from human donors can beconstructed and antibodies to a diverse array of antigens (includingself-antigens) can be isolated essentially following the techniquesdescribed by Mark et al., J. Mol. Biol. 222:581-597, 1991, or Griffithet al., EMBO J. 12:725-734, 1993. In a natural immune response, antibodygenes accumulate mutations at a high rate (somatic hypermutation). Someof the changes introduced will confer higher affinity, and B cellsdisplaying high-affinity surface immunoglobulin are preferentiallyreplicated and differentiated during subsequent antigen challenge. Thisnatural process can be mimicked by employing the technique known as“chain shuffling.” (Marks et al., Bio/Technol. 10:779-783, 1992). Inthis method, the affinity of “primary” human antibodies obtained byphage display can be improved by sequentially replacing the heavy andlight chain V region genes with repertoires of naturally occurringvariants (repertoires) of V domain genes obtained from unimmunizeddonors. This technique allows the production of antibodies and antibodyfragments with affinities in the pM-nM range. A strategy for making verylarge phage antibody repertoires (also known as “the mother-of-alllibraries”) has been described by Waterhouse et al., Nucl. Acids Res.21:2265-2266, 1993. Gene shuffling can also be used to derive humanantibodies from rodent antibodies, where the human antibody has similaraffinities and specificities to the starting rodent antibody. Accordingto this method, which is also referred to as “epitope imprinting”, theheavy or light chain V domain gene of rodent antibodies obtained byphage display technique is replaced with a repertoire of human V domaingenes, creating rodent-human chimeras. Selection on antigen results inisolation of human variable regions capable of restoring a functionalantigen-binding site, i.e., the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT Publication No.WO 93/06213). Unlike traditional humanization of rodent antibodies byCDR grafting, this technique provides completely human antibodies, whichhave no framework or CDR residues of rodent origin.

In some embodiments, antibodies may be made using hybridoma technology.It is contemplated that any mammalia subject including humans orantibody producing cells therefrom can be manipulated to serve as thebasis for production of mammalian, including human, hybridoma celllines. The route and schedule of immunization of the host animal aregenerally in keeping with established and conventional techniques forantibody stimulation and production, as further described herein.Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., 1975, Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the PD-1 monoclonal antibodies of the subjectinvention. The hybridomas or other immortalized B-cells are expanded andsubcloned, if desired, and supernatants are assayed for anti-immunogenactivity by conventional immunoassay procedures (e.g., radioimmunoassay,enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies specific for PD-1, or a portion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a PD-1 polypeptide, or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, the anti-PD-1 antagonist antibody (monoclonal or polyclonal)of interest may be sequenced and the polynucleotide sequence may then becloned into a vector for expression or propagation. The sequenceencoding the antibody of interest may be maintained in vector in a hostcell and the host cell can then be expanded and frozen for future use.Production of recombinant monoclonal antibodies in cell culture can becarried out through cloning of antibody genes from B cells by meansknown in the art. See, e.g. Tiller et al., 2008, J. Immunol. Methods329, 112; U.S. Pat. No. 7,314,622.

In some embodiments, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. Antibodies may also be customizedfor use, for example, in dogs, cats, primate, equines and bovines.

In some embodiments, fully human antibodies may be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are Xenomouse™ fromAbgenix, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.).

Antibodies may be made recombinantly by first isolating the antibodiesand antibody producing cells from host animals, obtaining the genesequence, and using the gene sequence to express the antibodyrecombinantly in host cells (e.g., CHO cells). Another method which maybe employed is to express the antibody sequence in plants (e.g.,tobacco) or transgenic milk. Methods for expressing antibodiesrecombinantly in plants or milk have been disclosed. See, for example,Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int.Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods231:147, 1999. Methods for making derivatives of antibodies, e.g.,domain, single chain, etc. are known in the art.

Immunoassays and flow cytometry sorting techniques such as fluorescenceactivated cell sorting (FACS) can also be employed to isolate antibodiesthat are specific for PD-1.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the monoclonal antibodies). The hybridoma cells serve asa preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors (such as expression vectors disclosed in PCTPublication No. WO 87/04462), which are then transfected into host cellssuch as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. See, e.g., PCT Publication No. WO 87/04462. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci.81:6851, 1984, or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In that manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of a PD-1 monoclonal antibodyherein.

Antibody fragments can be produced by proteolytic or other degradationof the antibodies, by recombinant methods (i.e., single or fusionpolypeptides) as described above or by chemical synthesis. Polypeptidesof the antibodies, especially shorter polypeptides up to about 50 aminoacids, are conveniently made by chemical synthesis. Methods of chemicalsynthesis are known in the art and are commercially available. Forexample, an antibody could be produced by an automated polypeptidesynthesizer employing the solid phase method. See also, U.S. Pat. Nos.5,807,715; 4,816,567; and 6,331,415.

In some embodiments, a polynucleotide comprises a sequence encoding theheavy chain and/or the light chain variable regions of antibody mAb1,mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9, mAb10, mAb11, mAb12,mAb13, mAb14, mAb15, or mAb16. The sequence encoding the antibody ofinterest may be maintained in a vector in a host cell and the host cellcan then be expanded and frozen for future use. Vectors (includingexpression vectors) and host cells are further described herein.

The invention includes affinity matured embodiments. For example,affinity matured antibodies can be produced by procedures known in theart (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al.,1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene,169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson etal., 1995, J. Immunol., 154(7):3310-9; Hawkins et al., 1992, J. Mol.Biol., 226:889-896; and PCT Publication No. WO2004/058184).

The following methods may be used for adjusting the affinity of anantibody and for characterizing a CDR. One way of characterizing a CDRof an antibody and/or altering (such as improving) the binding affinityof a polypeptide, such as an antibody, termed “library scanningmutagenesis”. Generally, library scanning mutagenesis works as follows.One or more amino acid positions in the CDR are replaced with two ormore (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20) amino acids using art recognized methods. This generatessmall libraries of clones (in some embodiments, one for every amino acidposition that is analyzed), each with a complexity of two or moremembers (if two or more amino acids are substituted at every position).Generally, the library also includes a clone comprising the native(unsubstituted) amino acid. A small number of clones, e.g., about 20-80clones (depending on the complexity of the library), from each libraryare screened for binding affinity to the target polypeptide (or otherbinding target), and candidates with increased, the same, decreased, orno binding are identified. Methods for determining binding affinity arewell-known in the art. Binding affinity may be determined using, forexample, Biacore™ surface plasmon resonance analysis, which detectsdifferences in binding affinity of about 2-fold or greater, Kinexa®Biosensor, scintillation proximity assays, ELISA, ORIGEN® immunoassay,fluorescence quenching, fluorescence transfer, and/or yeast display.Binding affinity may also be screened using a suitable bioassay.Biacore™ is particularly useful when the starting antibody already bindswith a relatively high affinity, for example a K_(D) of about 10 nM orlower.

In some embodiments, every amino acid position in a CDR is replaced (insome embodiments, one at a time) with all 20 natural amino acids usingart recognized mutagenesis methods (some of which are described herein).This generates small libraries of clones (in some embodiments, one forevery amino acid position that is analyzed), each with a complexity of20 members (if all 20 amino acids are substituted at every position).

In some embodiments, the library to be screened comprises substitutionsin two or more positions, which may be in the same CDR or in two or moreCDRs. Thus, the library may comprise substitutions in two or morepositions in one CDR. The library may comprise substitution in two ormore positions in two or more CDRs. The library may comprisesubstitution in 3, 4, 5, or more positions, said positions found in two,three, four, five or six CDRs. The substitution may be prepared usinglow redundancy codons. See, e.g., Table 2 of Balint et al., 1993, Gene137(1):109-18.

The CDR may be heavy chain variable region (VH) CDR3 and/or light chainvariable region (VL) CDR3. The CDR may be one or more of VH CDR1, VHCDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3. The CDR may be a KabatCDR, a Chothia CDR, an extended CDR, an AbM CDR, a contact CDR, or aconformational CDR.

Candidates with improved binding may be sequenced, thereby identifying aCDR substitution mutant which results in improved affinity (also termedan “improved” substitution). Candidates that bind may also be sequenced,thereby identifying a CDR substitution which retains binding.

Multiple rounds of screening may be conducted. For example, candidates(each comprising an amino acid substitution at one or more position ofone or more CDR) with improved binding are also useful for the design ofa second library containing at least the original and substituted aminoacid at each improved CDR position (i.e., amino acid position in the CDRat which a substitution mutant showed improved binding). Preparation,and screening or selection of this library is discussed further below.

Library scanning mutagenesis also provides a means for characterizing aCDR, in so far as the frequency of clones with improved binding, thesame binding, decreased binding or no binding also provide informationrelating to the importance of each amino acid position for the stabilityof the antibody-antigen complex. For example, if a position of the CDRretains binding when changed to all 20 amino acids, that position isidentified as a position that is unlikely to be required for antigenbinding. Conversely, if a position of CDR retains binding in only asmall percentage of substitutions, that position is identified as aposition that is important to CDR function. Thus, the library scanningmutagenesis methods generate information regarding positions in the CDRsthat can be changed to many different amino acids (including all 20amino acids), and positions in the CDRs which cannot be changed or whichcan only be changed to a few amino acids.

Candidates with improved affinity may be combined in a second library,which includes the improved amino acid, the original amino acid at thatposition, and may further include additional substitutions at thatposition, depending on the complexity of the library that is desired, orpermitted using the desired screening or selection method. In addition,if desired, adjacent amino acid position can be randomized to at leasttwo or more amino acids. Randomization of adjacent amino acids maypermit additional conformational flexibility in the mutant CDR, whichmay in turn, permit or facilitate the introduction of a larger number ofimproving mutations. The library may also comprise substitution atpositions that did not show improved affinity in the first round ofscreening.

The second library is screened or selected for library members withimproved and/or altered binding affinity using any method known in theart, including screening using Kinexa™ biosensor analysis, and selectionusing any method known in the art for selection, including phagedisplay, yeast display, and ribosome display.

To express the anti-PD-1 antibodies of the present invention, DNAfragments encoding VH and VL regions can first be obtained using any ofthe methods described above. Various modifications, e.g. mutations,deletions, and/or additions can also be introduced into the DNAsequences using standard methods known to those of skill in the art. Forexample, mutagenesis can be carried out using standard methods, such asPCR-mediated mutagenesis, in which the mutated nucleotides areincorporated into the PCR primers such that the PCR product contains thedesired mutations or site-directed mutagenesis.

The invention encompasses modifications to the variable regions shown inTable 1 and the CDRs shown in Tables 2, 3 or 4. For example, theinvention includes antibodies comprising functionally equivalentvariable regions and CDRs which do not significantly affect theirproperties as well as variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence may be mutated toobtain an antibody with the desired binding affinity to PD-1.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but framework alterations are alsocontemplated. Conservative substitutions are shown in Table 5 under theheading of “conservative substitutions.” If such substitutions result ina change in biological activity, then more substantial changes,denominated “exemplary substitutions” in Table 5, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

TABLE 5 Amino Acid Substitutions Original Conservative ResidueSubstitutions Exemplary Substitutions Ala (A) Val Val; Leu; Ile Arg (R)Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu;Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly(G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met;Ala: Phe; Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val;Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W)Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met;Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a β-sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) Non-polar Norleucine, Met, Ala, Val, Leu, lie;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gin;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

One type of substitution, for example, that may be made is to change oneor more cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. Forexample, there can be a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant region of an antibody. In some embodiments,the cysteine is canonical. Any cysteine residue not involved inmaintaining the proper conformation of the antibody also may besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant cross-linking. Conversely, cysteinebond(s) may be added to the antibody to improve its stability,particularly where the antibody is an antibody fragment such as an Fvfragment.

The antibodies may also be modified, e.g. in the variable domains of theheavy and/or light chains, e.g., to alter a binding property of theantibody. Changes in the variable region can alter binding affinityand/or specificity. In some embodiments, no more than one to fiveconservative amino acid substitutions are made within a CDR domain. Inother embodiments, no more than one to three conservative amino acidsubstitutions are made within a CDR domain. For example, a mutation maybe made in one or more of the CDR regions to increase or decrease theK_(D) Of the antibody for PD-1, to increase or decrease k_(off), or toalter the binding specificity of the antibody. Techniques insite-directed mutagenesis are well-known in the art. See, e.g., Sambrooket al. and Ausubel et al., supra.

A modification or mutation may also be made in a framework region orconstant region to increase the half-life of an anti-PD-1 antibody. See,e.g., PCT Publication No. WO 00/09560. A mutation in a framework regionor constant region can also be made to alter the immunogenicity of theantibody, to provide a site for covalent or non-covalent binding toanother molecule, or to alter such properties as complement fixation,FcR binding and antibody-dependent cell-mediated cytotoxicity. In someembodiments, no more than one to five conservative amino acidsubstitutions are made within the framework region or constant region.In other embodiments, no more than one to three conservative amino acidsubstitutions are made within the framework region or constant region.According to the invention, a single antibody may have mutations in anyone or more of the CDRs or framework regions of the variable domain orin the constant region.

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular,antibodies produced by CHO cells with tetracycline-regulated expressionof 1(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, NatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g. antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g. Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261 and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example, usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

Other methods of modification include using coupling techniques known inthe art, including, but not limited to, enzymatic means, oxidativesubstitution and chelation. Modifications can be used, for example, forattachment of labels for immunoassay. Modified polypeptides are madeusing established procedures in the art and can be screened usingstandard assays known in the art, some of which are described below andin the Examples.

In some embodiments, the Fc can be human IgG₂ or human IgG₄. In someembodiments, the antibody comprises a constant region of IgG₄ comprisingthe following mutations (Armour et al., 2003, Molecular Immunology 40585-593): E233F234L235 to P233V234A235 (IgG_(4Δc)), in which thenumbering is with reference to wild type IgG₄. In yet anotherembodiment, the Fc is human IgG₄ E233F234L235 to P233V234A235 withdeletion G236 (IgG_(4Δb)). In some embodiments the Fc is any human IgG₄Fc (IgG₄, IgG_(4Δb) or IgG_(4Δc)) containing hinge stabilizing mutationS228 to P228 (Aalberse et al., 2002, Immunology 105, 9-19). In otherembodiments, the Fc can be human IgG₂ containing the mutation A330P331to S330S331 (IgG_(2Δa)), in which the amino acid residues are numberedwith reference to the wild type IgG₂ sequence. Eur. J. Immunol., 1999,29:2613-2624.

In some embodiments, the antibody comprises a modified constant regionthat has increased or decreased binding affinity to a human Fc gammareceptor, is immunologically inert or partially inert, e.g., does nottrigger complement mediated lysis, does not stimulate antibody-dependentcell mediated cytotoxicity (ADCC), or does not activate microglia; orhas reduced activities (compared to the unmodified antibody) in any oneor more of the following: triggering complement mediated lysis,stimulating ADCC, or activating microglia. Different modifications ofthe constant region may be used to achieve optimal level and/orcombination of effector functions. See, for example, Morgan et al.,Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000;Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis et al.,Immunological Reviews 163:59-76, 1998. In some embodiments, the constantregion is modified as described in Eur. J. Immunol., 1999, 29:2613-2624;PCT Publication No. WO99/058572.

In some embodiments, an antibody constant region can be modified toavoid interaction with Fc gamma receptor and the complement and immunesystems. The techniques for preparation of such antibodies are describedin WO 99/58572. For example, the constant region may be engineered tomore resemble human constant regions to avoid immune response if theantibody is used in clinical trials and treatments in humans. See, e.g.,U.S. Pat. Nos. 5,997,867 and 5,866,692.

In still other embodiments, the constant region is aglycosylated forN-linked glycosylation. In some embodiments, the constant region isaglycosylated for N-linked glycosylation by mutating the oligosaccharideattachment residue and/or flanking residues that are part of theN-glycosylation recognition sequence in the constant region. Forexample, N-glycosylation site N297 may be mutated to, e.g., A, Q, K, orH. See, Tao et al., J. Immunology 143: 2595-2601, 1989; and Jefferis etal., Immunological Reviews 163:59-76, 1998. In some embodiments, theconstant region is aglycosylated for N-linked glycosylation. Theconstant region may be aglycosylated for N-linked glycosylationenzymatically (such as removing carbohydrate by enzyme PNGase), or byexpression in a glycosylation deficient host cell.

Other antibody modifications include antibodies that have been modifiedas described in PCT Publication No. WO 99/58572. These antibodiescomprise, in addition to a binding domain directed at the targetmolecule, an effector domain having an amino acid sequence substantiallyhomologous to all or part of a constant region of a human immunoglobulinheavy chain. These antibodies are capable of binding the target moleculewithout triggering significant complement dependent lysis, orcell-mediated destruction of the target. In some embodiments, theeffector domain is capable of specifically binding FcRn and/or FcγRIIb.These are typically based on chimeric domains derived from two or morehuman immunoglobulin heavy chain CH2 domains. Antibodies modified inthis manner are particularly suitable for use in chronic antibodytherapy, to avoid inflammatory and other adverse reactions toconventional antibody therapy.

In some embodiments, the antibody comprises a modified constant regionthat has increased binding affinity for FcRn and/or an increased serumhalf-life as compared with the unmodified antibody.

In a process known as “germlining”, certain amino acids in the VH and VLsequences can be mutated to match those found naturally in germline VHand VL sequences. In particular, the amino acid sequences of theframework regions in the VH and VL sequences can be mutated to match thegermline sequences to reduce the risk of immunogenicity when theantibody is administered. Germline DNA sequences for human VH and VLgenes are known in the art (see e.g., the “Vbase” human germlinesequence database; see also Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 227:776-798; and Cox et al., 1994, Eur. J.Immunol. 24:827-836).

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant regionof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues. Inanother example, the C-terminal lysine of the heavy chain of ananti-PD-1 antibody of the invention can be cleaved. In variousembodiments of the invention, the heavy and light chains of theanti-PD-1 antibodies may optionally include a signal sequence.

Once DNA fragments encoding the VH and VL segments of the presentinvention are obtained, these DNA fragments can be further manipulatedby standard recombinant DNA techniques, for example to convert thevariable region genes to full-length antibody chain genes, to Fabfragment genes, or to a scFv gene. In these manipulations, a VL- orVH-encoding DNA fragment is operatively linked to another DNA fragmentencoding another protein, such as an antibody constant region or aflexible linker. The term “operatively linked”, as used in this context,is intended to mean that the two DNA fragments are joined such that theamino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification.

The heavy chain constant region can be an IgG₁, IgG₂, IgG₃, IgG₄, IgA,IgE, IgM or IgD constant region, but most preferably is an IgG, or IgG₂constant region. The IgG constant region sequence can be any of thevarious alleles or allotypes known to occur among different individuals,such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypes representnaturally occurring amino acid substitution in the IgG1 constantregions. For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperatively linked to another DNA molecule encoding only the heavy chainCH1 constant region. The CH1 heavy chain constant region may be derivedfrom any of the heavy chain genes.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region. The kappa constant region maybe any of the various alleles known to occur among differentindividuals, such as Inv(1), Inv(2), and Inv(3). The lambda constantregion may be derived from any of the three lambda genes.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker suchthat the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (See e.g., Bird et al., 1988, Science 242:423-426; Huston et al.,1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990,Nature 348:552-554. An example of a linking peptide is (GGGGS)₃ (SEQ IDNO: 19), which bridges approximately 3.5 nm between the carboxy terminusof one variable region and the amino terminus of the other variableregion. Linkers of other sequences have been designed and used (Bird etal., 1988, supra). Linkers can in turn be modified for additionalfunctions, such as attachment of drugs or attachment to solid supports.The single chain antibody may be monovalent, if only a single VH and VLare used, bivalent, if two VH and VL are used, or polyvalent, if morethan two VH and VL are used. Bispecific or polyvalent antibodies may begenerated that bind specifically to PD-1 and to another molecule. Thesingle chain variants can be produced either recombinantly orsynthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

Other forms of single chain antibodies, such as diabodies, are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL are expressed on a single polypeptide chain, but using a linkerthat is too short to allow for pairing between the two domains on thesame chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al., 1993, Proc. Natl. Acad Sci. USA90:6444-6448; Poljak, R. J., et al., 1994, Structure 2:1121-1123).

Heteroconjugate antibodies, comprising two covalently joined antibodies,are also within the scope of the invention. Such antibodies have beenused to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (PCT Publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents and techniques are well known in the art, and are described inU.S. Pat. No. 4,676,980.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods of synthetic protein chemistry, including those involvingcross-linking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

The invention also encompasses fusion proteins comprising one or morefragments or regions from the antibodies disclosed herein. In someembodiments, a fusion antibody may be made that comprises all or aportion of an anti-PD-1 antibody of the invention linked to anotherpolypeptide. In another embodiment, only the variable domains of theanti-PD-1 antibody are linked to the polypeptide. In another embodiment,the VH domain of an anti-PD-1 antibody is linked to a first polypeptide,while the VL domain of an anti-PD-1 antibody is linked to a secondpolypeptide that associates with the first polypeptide in a manner suchthat the VH and VL domains can interact with one another to form anantigen binding site. In another preferred embodiment, the VH domain isseparated from the VL domain by a linker such that the VH and VL domainscan interact with one another. The VH-linker-VL antibody is then linkedto the polypeptide of interest. In addition, fusion antibodies can becreated in which two (or more) single-chain antibodies are linked to oneanother. This is useful if one wants to create a divalent or polyvalentantibody on a single polypeptide chain, or if one wants to create abispecific antibody.

In some embodiments, a fusion polypeptide is provided that comprises atleast 10 contiguous amino acids of the variable light chain region shownin SEQ ID NO: 2, 7, 8, or 9 and/or at least 10 amino acids of thevariable heavy chain region shown in SEQ ID NO: 3, 4, 5, or 6. In otherembodiments, a fusion polypeptide is provided that comprises at leastabout 10, at least about 15, at least about 20, at least about 25, or atleast about 30 contiguous amino acids of the variable light chain regionand/or at least about 10, at least about 15, at least about 20, at leastabout 25, or at least about 30 contiguous amino acids of the variableheavy chain region. In another embodiment, the fusion polypeptidecomprises a light chain variable region and/or a heavy chain variableregion, as shown in any of the sequence pairs selected from among SEQ IDNOs: 2 and 3, 7 and 3, 8 and 3, 8 9 and 3, 2 and 4, 7 and 4, 8 and 4, 9and 4, 2 and 5, 7 and 5, 8 and 5, 9 and 5, 2 and 6, 7 and 6, 8 and 6,and 9 and 6. In another embodiment, the fusion polypeptide comprises oneor more CDR(s). In still other embodiments, the fusion polypeptidecomprises VH CDR3 and/or VL CDR3. For purposes of this invention, afusion protein contains one or more antibodies and another amino acidsequence to which it is not attached in the native molecule, forexample, a heterologous sequence or a homologous sequence from anotherregion. Exemplary heterologous sequences include, but are not limited toa “tag” such as a FLAG tag or a 6His tag. Tags are well known in theart.

A fusion polypeptide can be created by methods known in the art, forexample, synthetically or recombinantly. Typically, the fusion proteinsof this invention are made by preparing and expressing a polynucleotideencoding them using recombinant methods described herein, although theymay also be prepared by other means known in the art, including, forexample, chemical synthesis.

In other embodiments, other modified antibodies may be prepared usinganti-PD-1 antibody encoding nucleic acid molecules. For instance, “Kappabodies” (III et al., 1997, Protein Eng. 10:949-57), “Minibodies” (Martinet al., 1994, EMBO J. 13:5303-9), “Diabodies” (Holliger et al., supra),or “Janusins” (Traunecker et al., 1991, EMBO J. 10:3655-3659 andTraunecker et al., 1992, Int. J. Cancer (Suppl.) 7:51-52) may beprepared using standard molecular biological techniques following theteachings of the specification.

For example, bispecific antibodies, monoclonal antibodies that havebinding specificities for at least two different antigens, can beprepared using the antibodies disclosed herein. Methods for makingbispecific antibodies are known in the art (see, e.g., Suresh et al.,1986, Methods in Enzymology 121:210). For example, bispecific antibodiesor antigen-binding fragments can be produced by fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, 1990,Clin. Exp. Immunol. 79:315-321, Kostelny et al., 1992, J. Immunol.148:1547-1553. Traditionally, the recombinant production of bispecificantibodies was based on the coexpression of two immunoglobulin heavychain-light chain pairs, with the two heavy chains having differentspecificities (Millstein and Cuello, 1983, Nature 305, 537-539). Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some embodiments, the bispecific antibody binds to twodifferent epitopes of PD-1. In some embodiments, the modified antibodiesdescribed above are prepared using one or more of the variable domainsor CDR regions from an anti-PD-1 antibody provided herein.

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantregion sequences. The fusion preferably is with an immunoglobulin heavychain constant region, comprising at least part of the hinge, CH2 andCH3 regions. It is preferred to have the first heavy chain constantregion (CH1), containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are cotransfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one approach, the bispecific antibodies are composed of a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structure,with an immunoglobulin light chain in only one half of the bispecificmolecule, facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations. This approach isdescribed in PCT Publication No. WO 94/04690.

This invention also provides compositions comprising antibodiesconjugated (for example, linked) to an agent that facilitate coupling toa solid support (such as biotin or avidin). For simplicity, referencewill be made generally to antibodies with the understanding that thesemethods apply to any of the PD-1 binding and/or antagonist embodimentsdescribed herein. Conjugation generally refers to linking thesecomponents as described herein. The linking (which is generally fixingthese components in proximate association at least for administration)can be achieved in any number of ways. For example, a direct reactionbetween an agent and an antibody is possible when each possesses asubstituent capable of reacting with the other. For example, anucleophilic group, such as an amino or sulfhydryl group, on one may becapable of reacting with a carbonyl-containing group, such as ananhydride or an acid halide, or with an alkyl group containing a goodleaving group (e.g., a halide) on the other.

The antibodies can be bound to many different carriers. Carriers can beactive and/or inert. Examples of well-known carriers includepolypropylene, polystyrene, polyethylene, dextran, nylon, amylases,glass, natural and modified celluloses, polyacrylamides, agaroses andmagnetite. The nature of the carrier can be either soluble or insolublefor purposes of the invention. Those skilled in the art will know ofother suitable carriers for binding antibodies, or will be able toascertain such, using routine experimentation. In some embodiments, thecarrier comprises a moiety that targets the lung, heart, or heart valve.

An antibody or polypeptide of this invention may be linked to a labelingagent such as a fluorescent molecule, a radioactive molecule or anyothers labels known in the art. Labels are known in the art whichgenerally provide (either directly or indirectly) a signal.

Polynucleotides, Vectors, and Host Cells

The invention also provides polynucleotides encoding any of theantibodies, including antibody fragments and modified antibodiesdescribed herein, such as, e.g., antibodies having impaired effectorfunction. In another aspect, the invention provides a method of makingany of the polynucleotides described herein. Polynucleotides can be madeand expressed by procedures known in the art. Accordingly, the inventionprovides polynucleotides or compositions, including pharmaceuticalcompositions, comprising polynucleotides, encoding any of the following:the antibodies mAb1, mAb2, mAb3, mAb4, mAb5, mAb6, mAb7, mAb8, mAb9,mAb10, mAb11, mAb12, mAb13, mAb14, mAb15, mAb16, and mAb17 or anyfragment or part thereof having the ability to antagonize PD-1.

Polynucleotides complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a fragment thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a fragmentthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegAlign® program in the Lasergene® suite of bioinformatics software(DNASTAR®, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors are further provided. Expression vectors generallyare replicable polynucleotide constructs that contain a polynucleotideaccording to the invention. It is implied that an expression vector mustbe replicable in the host cells either as episomes or as an integralpart of the chromosomal DNA. Suitable expression vectors include but arenot limited to plasmids, viral vectors, including adenoviruses,adeno-associated viruses, retroviruses, cosmids, and expressionvector(s) disclosed in PCT Publication No. WO 87/04462. Vectorcomponents may generally include, but are not limited to, one or more ofthe following: a signal sequence; an origin of replication; one or moremarker genes; suitable transcriptional controlling elements (such aspromoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

The invention also provides host cells comprising any of thepolynucleotides described herein. Any host cells capable ofover-expressing heterologous DNAs can be used for the purpose ofisolating the genes encoding the antibody, polypeptide or protein ofinterest. Non-limiting examples of mammalian host cells include but notlimited to COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes (such asE. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; orK. lactis). Preferably, the host cells express the cDNAs at a level ofabout 5 fold higher, more preferably, 10 fold higher, even morepreferably, 20 fold higher than that of the corresponding endogenousantibody or protein of interest, if present, in the host cells.Screening the host cells for a specific binding to PD-1 or a PD-1 domainis effected by an immunoassay or FACS. A cell overexpressing theantibody or protein of interest can be identified.

An expression vector can be used to direct expression of an anti-PD-1antagonist antibody. One skilled in the art is familiar withadministration of expression vectors to obtain expression of anexogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908;6,413,942; and 6,376,471. Administration of expression vectors includeslocal or systemic administration, including injection, oraladministration, particle gun or catheterized administration, and topicaladministration. In another embodiment, the expression vector isadministered directly to the sympathetic trunk or ganglion, or into acoronary artery, atrium, ventrical, or pericardium.

Targeted delivery of therapeutic compositions containing an expressionvector, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described in, for example,Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., GeneTherapeutics: Methods And Applications Of Direct Gene Transfer, J. A.Wolff, ed., 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al.,J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA,1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeuticcompositions containing a polynucleotide are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. Concentration ranges of about 500 ng to about 50 mg,about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg toabout 100 μg of DNA can also be used during a gene therapy protocol. Thetherapeutic polynucleotides and polypeptides can be delivered using genedelivery vehicles. The gene delivery vehicle can be of viral ornon-viral origin (see generally, Jolly, Cancer Gene Therapy, 1994, 1:51;Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy,1995, 1:185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression ofsuch coding sequences can be induced using endogenous mammalian orheterologous promoters. Expression of the coding sequence can be eitherconstitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther., 1992, 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther., 1992,3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 1989,264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additionalapproaches are described in Philip, Mol. Cell Biol., 1994, 14:2411, andin Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

Compositions

The invention also provides pharmaceutical compositions comprising aneffective amount of an anti-PD-1 antibody described herein. Examples ofsuch compositions, as well as how to formulate, are also describedherein. In some embodiments, the composition comprises one or more PD-1antibodies. In other embodiments, the anti-PD-1 antibody recognizesPD-1. In other embodiments, the anti-PD-1 antibody is a human antibody.In other embodiments, the anti-PD-1 antibody is a humanized antibody. Insome embodiments, the anti-PD-1 antibody comprises a constant regionthat is capable of triggering a desired immune response, such asantibody-mediated lysis or ADCC. In other embodiments, the anti-PD-1antibody comprises a constant region that does not trigger an unwantedor undesirable immune response, such as antibody-mediated lysis or ADCC.In other embodiments, the anti-PD-1 antibody comprises one or moreCDR(s) of the antibody (such as one, two, three, four, five, or, in someembodiments, all six CDRs).

It is understood that the compositions can comprise more than oneanti-PD-1 antibody (e.g., a mixture of PD-1 antibodies that recognizedifferent epitopes of PD-1). Other exemplary compositions comprise morethan one anti-PD-1 antibody that recognize the same epitope(s), ordifferent species of anti-PD-1 antibodies that bind to differentepitopes of PD-1. In some embodiments, the compositions comprise amixture of anti-PD-1 antibodies that recognize different variants ofPD-1.

The composition used in the present invention can further comprisepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and practice of Pharmacy 20th Ed., 2000,Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations, and may comprise buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Pharmaceutically acceptable excipients arefurther described herein.

The anti-PD-1 antibody and compositions thereof can also be used inconjunction with, or administered separately, simultaneously, orsequentially with other agents that serve to enhance and/or complementthe effectiveness of the agents.

The invention also provides compositions, including pharmaceuticalcompositions, comprising any of the polynucleotides of the invention. Insome embodiments, the composition comprises an expression vectorcomprising a polynucleotide encoding the antibody as described herein.In other embodiment, the composition comprises an expression vectorcomprising a polynucleotide encoding any of the antibodies describedherein.

Methods for Preventing or Treating Conditions Mediated by PD-1

The antibodies and the antibody conjugates of the present invention areuseful in various applications including, but are not limited to,therapeutic treatment methods and diagnostic treatment methods.

In one aspect, the invention provides a method for treating a cancer. Insome embodiments, the method of treating a cancer in a subject comprisesadministering to the subject in need thereof an effective amount of acomposition (e.g., pharmaceutical composition) comprising any of thePD-1 antibodies as described herein. As used herein, cancers include,but are not limited to bladder cancer, breast cancer, cervical cancer,choriocarcinoma, colon cancer, esophageal cancer, gastric cancer,glioblastoma, glioma, brain tumor, head and neck cancer, kidney cancer,lung cancer, oral cancer, ovarian cancer, pancreatic cancer, prostatecancer, liver cancer, uterine cancer, bone cancer, leukemia, lymphoma,sacrcoma, blood cancer, thyroid cancer, thymic cancer, eye cancer, andskin cancer. In some embodiments, provided is a method of inhibitingtumor growth or progression in a subject, comprising administering tothe subject in need thereof an effective amount of a compositioncomprising the PD-1 antibodies or the PD-1 antibody conjugates asdescribed herein. In some embodiments, the tumor is a PD-L1 expressingtumor. In other embodiments, the tumor does not express PD-L1. In otherembodiments, provided is a method of inhibiting metastasis of cancercells in a subject, comprising administering to the subject in needthereof an effective amount of a composition comprising any of the PD-1antibodies as described herein. In other embodiments, provided is amethod of inducing regression of a tumor in a subject, comprisingadministering to the subject in need thereof an effective amount of acomposition comprising any of the PD-1 antibodies as described herein.

In another aspect, provided is a method of detecting, diagnosing, and/ormonitoring a cancer. For example, the PD-1 antibodies as describedherein can be labeled with a detectable moiety such as an imaging agentand an enzyme-substrate label. The antibodies as described herein canalso be used for in vivo diagnostic assays, such as in vivo imaging(e.g., PET or SPECT), or a staining reagent.

In some embodiments, the methods described herein further comprise astep of treating a subject with an additional form of therapy. In someembodiments, the additional form of therapy is an additional anti-cancertherapy including, but not limited to, chemotherapy, radiation, surgery,hormone therapy, and/or additional immunotherapy.

With respect to all methods described herein, reference to anti-PD-1antagonist antibodies also includes compositions comprising one or moreadditional agents. These compositions may further comprise suitableexcipients, such as pharmaceutically acceptable excipients includingbuffers, which are well known in the art. The present invention can beused alone or in combination with other methods of treatment.

The anti-PD-1 antagonist antibody can be administered to a subject viaany suitable route. It should be apparent to a person skilled in the artthat the examples described herein are not intended to be limiting butto be illustrative of the techniques available. Accordingly, in someembodiments, the anti-PD-1 antagonist antibody is administered to asubject in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerebrospinal,transdermal, subcutaneous, intra-articular, sublingually, intrasynovial,via insufflation, intrathecal, oral, inhalation or topical routes.Administration can be systemic, e.g., intravenous administration, orlocalized. Commercially available nebulizers for liquid formulations,including jet nebulizers and ultrasonic nebulizers are useful foradministration. Liquid formulations can be directly nebulized andlyophilized powder can be nebulized after reconstitution. Alternatively,anti-PD-1 antagonist antibody can be aerosolized using a fluorocarbonformulation and a metered dose inhaler, or inhaled as a lyophilized andmilled powder.

In some embodiments, an anti-PD-1 antagonist antibody is administeredvia site-specific or targeted local delivery techniques. Examples ofsite-specific or targeted local delivery techniques include variousimplantable depot sources of the anti-PD-1 antagonist antibody or localdelivery catheters, such as infusion catheters, indwelling catheters, orneedle catheters, synthetic grafts, adventitial wraps, shunts and stentsor other implantable devices, site specific carriers, direct injection,or direct application. See, e.g., PCT Publication No. WO 00/53211 andU.S. Pat. No. 5,981,568.

Various formulations of an anti-PD-1 antagonist antibody may be used foradministration. In some embodiments, the anti-PD-1 antagonist antibodymay be administered neat. In some embodiments, anti-PD-1 antagonistantibody and a pharmaceutically acceptable excipient may be in variousformulations. Pharmaceutically acceptable excipients are known in theart, and are relatively inert substances that facilitate administrationof a pharmacologically effective substance. For example, an excipientcan give form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000.

In some embodiments, these agents are formulated for administration byinjection (e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). Accordingly, these agents can be combined withpharmaceutically acceptable vehicles such as saline, Ringer's solution,dextrose solution, and the like. The particular dosage regimen, i.e.,dose, timing and repetition, will depend on the particular individualand that individual's medical history.

An anti-PD-1 antagonist antibody can be administered using any suitablemethod, including by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Anti-PD-1 antibodies can also beadministered topically or via inhalation, as described herein.Generally, for administration of anti-PD-1 antibodies, an initialcandidate dosage can be about 2 mg/kg. For the purpose of the presentinvention, a typical daily dosage might range from about any of 3 μg/kgto 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg, to 100 mg/kg or more,depending on the factors mentioned above. For example, dosage of about 1mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25mg/kg may be used. For repeated administrations over several days orlonger, depending on the condition, the treatment is sustained until adesired suppression of symptoms occurs or until sufficient therapeuticlevels are achieved, for example, to reduce symptoms associated withcancer. The progress of this therapy is easily monitored by conventionaltechniques and assays. The dosing regimen (including the anti-PD-1antagonist antibody used) can vary over time.

For the purpose of the present invention, the appropriate dosage of ananti-PD-1 antagonist antibody will depend on the anti-PD-1 antagonistantibody (or compositions thereof) employed, the type and severity ofsymptoms to be treated, whether the agent is administered for preventiveor therapeutic purposes, previous therapy, the patients clinical historyand response to the agent, the patient's clearance rate for theadministered agent, and the discretion of the attending physician.Typically the clinician will administer an anti-PD-1 antagonist antibodyuntil a dosage is reached that achieves the desired result. Dose and/orfrequency can vary over course of treatment. Empirical considerations,such as the half-life, generally will contribute to the determination ofthe dosage. For example, antibodies that are compatible with the humanimmune system, such as humanized antibodies or fully human antibodies,may be used to prolong half-life of the antibody and to prevent theantibody being attacked by the host's immune system. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of symptoms.Alternatively, sustained continuous release formulations of anti-PD-1antagonist antibodies may be appropriate. Various formulations anddevices for achieving sustained release are known in the art.

In one embodiment, dosages for an antagonist antibody may be determinedempirically in individuals who have been given one or moreadministration(s) of an antagonist antibody. Individuals are givenincremental dosages of an anti-PD-1 antagonist antibody. To assessefficacy, an indicator of the disease can be followed.

Administration of an anti-PD-1 antagonist antibody in accordance withthe method in the present invention can be continuous or intermittent,depending, for example, upon the recipients physiological condition,whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an anti-PD-1 antagonist antibody may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced doses.

In some embodiments, more than one anti-PD-1 antagonist antibody may bepresent. At least one, at least two, at least three, at least four, atleast five different, or more antagonist antibodies can be present.Generally, those anti-PD-1 antagonist antibodies may have complementaryactivities that do not adversely affect each other. An anti-PD-1antagonist antibody can also be used in conjunction with otherantibodies and/or other therapies. An anti-PD-1 antagonist antibody canalso be used in conjunction with other agents that serve to enhanceand/or complement the effectiveness of the agents.

In some embodiments, the anti-PD-1 antagonist antibody may beadministered in combination with the administration of one or moreadditional therapeutic agents. These include, but are not limited to,the administration of a chemotherapeutic agent, a vaccine, a CAR-Tcell-based therapy, radiotherapy, a cytokine therapy, a vaccine, ananti-PD-1 bispecific antibody, an inhibitor of other immunosuppressivepathways, an inhibitors of angiogenesis, a T cell activator, aninhibitor of a metabolic pathway, an mTOR inhibitor, an inhibitor of anadenosine pathway, a tyrosine kinase inhibitor including but not limitedto inlyta, ALK inhibitors and sunitinib, a BRAF inhibitor, an epigeneticmodifier, an inhibitors or depletor of Treg cells and/or ofmyeloid-derived suppressor cells, a JAK inhibitor, a STAT inhibitor, acyclin-dependent kinase inhibitor, a biotherapeutic agent (including butnot limited to antibodies to VEGF, VEGFR, EGFR, Her2/neu, other growthfactor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, and ICOS),an immunogenic agent (for example, attenuated cancerous cells, tumorantigens, antigen presenting cells such as dendritic cells pulsed withtumor derived antigen or nucleic acids, immune stimulating cytokines(for example, IL-2, IFNα2, GM-CSF), and cells transfected with genesencoding immune stimulating cytokines such as but not limited toGM-CSF). Examples of chemotherapeutic agents include alkylating agentssuch as thiotepa and cyclosphosphamide; alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, especially calicheamicin gammall and calicheamicin phill,see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(including morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, and deoxydoxorubicin), pegylated liposomaldoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol;nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2, 2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;mitoxantrone; vincristine; vinorelbine; novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO);retinoids such as retinoic acid; capecitabine; and pharmaceuticallyacceptable salts, acids or derivatives of any of the above. Alsoincluded are anti-hormonal agents that act to regulate or inhibithormone action on tumors such as anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen,raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and toremifene (Fareston); aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole,vorozole, letrozole, and anastrozole; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

In some embodiments, an anti-PD-1 antagonist antibody is used inconjunction with one or more other therapeutic agents targeting animmune checkpoint modulator, such as, for example without limitation, anagent targeting PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC (PD-L2),B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47,CD80, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160,TIM1, TIM-3, TIM4, VISTA (PD-H1), OX40, OX40L, GITR, GITRL, CD70, CD27,4-1BB, 4-BBL, DR3, TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM(SLAMF1, CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF4 (2B4, CD244),SLAMF5 (CD84), SLAMF6 (NTB-A), SLAMCF7 (CS1), SLAMF8 (BLAME), SLAMF9(CD2F), CD28, CEACAM1(CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6,CEACAM7, CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1-15, PSG1-11,CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73-adenosinepathway (A2AR), BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, VEGF pathway,CSF-1, or an innate immune response modulator. In some embodiments, ananti-PD-1 antagonist antibody is used in conjunction with, for example,an anti-PD-L1 antagonist antibody such, as for example, BMS-936559(MDX-1105) and MPDL3280A; an anti-PD-1 antagonist antibody such as forexample, nivolumab, pembrolizumab, and pidilizumab; an anti-CTLA-4antagonist antibody such as for example ipilimumab; an anti-LAG-3antagonist antibody such as BMS-986016 and IMP701; an anti-TIM-3antagonist antibody; an anti-B7-H3 antagonist antibody such as forexample MGA271; an-anti-VISTA antagonist antibody; an anti-TIGITantagonist antibody; an anti-CD28 antagonist antibody; an anti-CD80antibody; an anti-CD86 antibody; an-anti-B7-H4 antagonist antibody; ananti-ICOS agonist antibody; an anti-CD28 agonist antibody; an innateimmune response modulator (e.g., TLRs, KIR, NKG2A), and an IDOinhibitor. In some embodiments, an anti-PD-1 antagonist antibody is usedin conjunction with a 4-1BB (CD137) agonist such as, for example,PF-05082566 or BMS-663513. In some embodiments, an anti-PD-1 antagonistantibody is used in conjunction with an OX40 agonist such as, forexample, an anti-OX-40 agonist antibody. In some embodiments, ananti-PD-1 antagonist antibody is used in conjunction with a GITR agonistsuch as, for example, an-anti-GITR agonist antibody such as, for examplewithout limitation, TRX518. In some embodiments, an anti-PD-1 antagonistantibody is used in conjunction with an IDO inhibitor. In someembodiments, an anti-PD-1 antagonist antibody is used in conjunctionwith a cytokine therapy such as, for example without limitation, IL-15,CSF-1, MCSF-1, etc.

In some embodiments, an anti-PD-1 antagonist antibody is used inconjunction with one or more other therapeutic antibodies, such as, forexample without limitation, an antibody targeting CD19, CD22, CD40,CD52, or CCR4.

In some embodiments, the anti-PD-1 antibody therapy may precede orfollow the other agent treatment by intervals ranging from minutes toweeks. In embodiments where the other agents and/or a proteins orpolynucleotides are administered separately, one would generally ensurethat a significant period of time did not expire between each delivery,such that the agent and the composition of the present invention wouldstill be able to exert an advantageously combined effect on the subject.In such instances, it is contemplated that one may administer bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other. In some situations, it may bedesirable to extend the time period for administration significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

In some embodiments, an anti-PD-1 antagonist antibody compositioncomprises a second agent selected from crizotinib, palbociclib,gemcitabine, cyclophosphamide, fluorouracil, FOLFOX, folinic acid,oxaliplatin, axitinib, sunitinib malate, tofacitinib, bevacizumab,rituximab, and traztuzumab.

In some embodiments, an anti-PD-1 antibody composition is combined witha treatment regimen further comprising a traditional therapy selectedfrom the group consisting of: surgery, radiation therapy, chemotherapy,targeted therapy, immunotherapy, hormonal therapy, angiogenesisinhibition and palliative care.

Use of the PD-1 Antibodies in Vaccine-Based Immunotherapy Regimens forCancer

In some particular embodiments, the present disclosure provides a methodfor enhancing the immunogenicity or therapeutic effect of a vaccine forthe treatment of a cancer in a mammal, particularly a human, whichmethod comprises administering to the mammal receiving the vaccine aneffective amount of anti-PD-1 antagonist antibody provided by thepresent disclosure. In some other particular embodiments, the presentdisclosure provides a method for treating a cancer in a mammal,particularly a human, which method comprises administering to the mammal(1) an effective amount of a vaccine capable of eliciting an immuneresponse against cells of the cancer and (2) an effective amount of ananti-PD-1 antagonist antibody provided by the present disclosure. Themethod of treating a neoplastic disorder in a mammal and the method ofenhancing the immunogenicity or therapeutic effect of a vaccine for thetreatment of a neoplastic disorder in a mammal described herein aboveare collectively referred to as “vaccine-based immunotherapy regimensfor cancer” (or “VBIR for cancer”).

In the VBIR for cancer, the vaccine may be in any form or formulations,such as (i) cell-based vaccines, (ii) subunit vaccines, (iii)protein-based vaccines, (iv) peptide-based vaccines, or (v) nucleicacid-based vaccines (such as DNA-based vaccines, RNA-based vaccines,plasmid-based vaccines, or viral vector-based vaccines).

The VBIR for cancer provided by the present disclosure may be applicablefor any type of cancers. Examples of specific cancers include:small-cell lung cancer, non-small cell lung cancer, glioma, gastriccancer, gastrointestinal cancer, renal cancer, ovarian cancer, livercancer, colorectal cancer, endometrial cancer, kidney cancer, prostatecancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastomamultiforme, cervical cancer, bladder cancer, breast cancer, and head andneck cancer.

Vaccines intended for treating cancers typically contain an antigen (inthe form of a peptide, protein, cell component, whole cell, or a nucleicacid molecule encoding a peptide-based antigen) that is capable ofeliciting an immune response against a particular TAA expressed on or bycells of the target tumor. Many TAAs are known in the art. Examples ofknown TAAs include: PSA, PSCA, and PSMA for prostate cancer, CEA, MUC-1,Ep-CAM, 5T4, hCG-b, K-ras, and TERT for colorectal cancer, CEA, Muc-1,p53, mesothelin, Survivin, and NY-ESO-1 for ovarian cancer; Muc-1, 5T4,WT-1, TERT, CEA, EGF-R and MAGE-A3 for non-small cell lung cancer; 5T4for renal cell carcinoma; and Muc-1, mesothelin, K-Ras, Annexin A2,TERT, and CEA for pancreatic cancer. In some particular embodiments, thevaccine used in the VBIR for cancer provided by the present disclosureis selected from the group consisting of:

(1) a vaccine capable of eliciting an immune response against a TAAselected from PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R, orMAGE-A3;

(2) a vaccine containing a peptide antigen derived from a TAA selectedfrom PSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R, or MAGE-A3;and

(3) a vaccine containing a nucleic acid molecule that encodes a peptideantigen, wherein the peptide antigen is derived from a TAA selected fromPSA, PSCA, PSMA, CEA, MUC-1, TERT, mesothelin, EGF-R, or MAGE-A3.

In still other particular embodiments, the vaccine contains a nucleicacid molecule that encodes one or more immunogenic polypeptides derivedfrom PSA, one or more immunogenic polypeptides derived from PSCA, or oneor more immunogenic polypeptides derived from PSMA.

In a specific embodiment, the nucleic acid molecule is selected from thegroup consisting of:

(1) a nucleic acid molecule encoding an immunogenic polypeptide derivedfrom the human PSMA of SEQ ID NO:42;

(2) a nucleic acid molecule encoding an immunogenic polypeptidecomprising amino acids 15-750 of SEQ ID NO:42;

(3) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 43, or a degenerate variant thereof;

(4) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 44, or a degenerate variant thereof;

(5) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 45, or a degenerate variant thereof;

(6) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 46, or a degenerate variant thereof;

(7) a nucleic acid molecule encoding an immunogenic polypeptide derivedfrom the human PSA of SEQ ID NO:47;

(8) a nucleic acid molecule encoding an immunogenic polypeptidecomprising amino acids 25-261 of SEQ ID NO:47;

(9) a nucleic acid molecule encoding an immunogenic polypeptide derivedfrom the human PSCA of SEQ ID NO:48;

(10) a nucleic acid molecule encoding (i) an immunogenic polypeptidederived from the human PSMA of SEQ ID NO:42, (ii) an immunogenicpolypeptide derived from the human PSA of SEQ ID NO:47, and (iii) animmunogenic polypeptide derived from the human PSCA of SEQ ID NO:48; and

(11) a nucleic acid molecule encoding (i) an immunogenic polypeptidecomprising amino acids 15-750 of SEQ ID NO:42, (ii) an immunogenicpolypeptide comprising amino acids 25-261 of SEQ ID NO:47, and (iii) animmunogenic polypeptide of SEQ ID NO:48.

The nucleic acid molecules that encode one or more immunogenicpolypeptides derived from prostate-associated antigens may be in theform of plasmids or vectors. An example of such a plasmid is the nucleicacid construct of SEQ ID NO:46 (also referred to as Plasmid 458). Thenucleotide sequence of a vector that expresses an immunogenicpolypeptide derived from human PSMA is set forth in SEQ ID NO:44 (alsoreferred to as vector AdC68W). The nucleotide sequence of a vector thatexpresses an immunogenic polypeptide derived from human PSMA, animmunogenic polypeptide derived from human PSA, and an immunogenicpolypeptide derived from human PSCA is set forth in SEQ ID NO:45 and avector human PSMA (vector AdC68W-734). Various immunogenic polypeptidesderived from human PSMA, PSA, and PSCA, nucleic acid constructs(including plasmids and vectors) encoding such immunogenic polypeptides,and methods for preparing the immunogenic polypeptides and nucleic acidconstructs, including Plasmid 458, and vector AdC68W and AdC68W-734, aredisclosed in Internationals Application Publications WO2013/164754 andWO 2015/063647, each of which is incorporated herein by reference in itsentirety.

In one aspect, the invention provides an isolated antagonist antibodywhich specifically binds to PD-1, wherein the antibody comprises a heavychain variable region (VH) comprising a VH complementarity determiningregion one (CDR1), VH CDR2, and VH CDR3 of the VH having an amino acidsequence selected group the group consisting of SEQ ID NO: 3, SEQ ID NO:4; SEQ ID NO: 5; and SEQ ID NO: 6; and a light chain variable region(VL) comprising a VL CDR1, VL CDR2, and VL CDR3 of the VL having anamino acid sequence selected from the group consisting of SEQ ID NO: 2;SEQ ID NO:7; SEQ ID NO: 8; and SEQ ID NO: 9.

Any anti-PD-1 antagonist antibodies disclosed in the present disclosuremay be used in the VBIR for cancer. In some embodiments, the anti-PD-1antagonist antibody comprises a VH region and/or a VL region, whereinthe VH region comprises the amino acid sequence shown in SEQ ID NO: 3,4, 5, or 6, or a variant with one or several conservative amino acidsubstitutions in residues that are not within a CDR, and wherein the VLregion comprises the amino acid sequence shown in SEQ ID NO: 2, 7, 8, or9, or a variant thereof with one or several amino acid substitutions inamino acids that are not within a CDR. In some embodiments, the antibodycomprises a light chain comprising the sequence shown in SEQ ID NO: 39and/or a heavy chain comprising the sequence shown in SEQ ID NO: 29 or38. In some particular embodiments, the antibody comprises a VH regionproduced by the expression vector with ATCC Accession No. PTA-121183. Insome embodiments, the antibody comprises a VL region produced by theexpression vector with ATCC Accession No. PTA-121182.

The VBIR for cancer provided by the present disclosure may furthercomprise one or more other immune modulators (in addition to the PD-1antagonist antibody provided by the present disclosure). The otherimmune modulators may be an immune-effector-cell enhancer (“IECenhancer”) or an immune-suppressive-cell inhibitor (“ISC inhibitor”).The additional IEC enhancer or additional ISC inhibitor may be usedalone in combination with the VBIR for cancer. The additional IECenhancer and additional ISC inhibitor may also be used together incombination with the VBIR for cancer.

Examples of classes of ISC inhibitors include protein kinase inhibitors,cyclooxygenase-2 (COX-2) inhibitors, phosphodiesterase type 5 (PDE5)inhibitors, and DNA crosslinkers. Examples of COX-2 inhibitors includecelecoxib and rofecoxib. Examples of PDE5 inhibitors include avanafil,lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, andzaprinast. An example of DNA crosslinkers is cyclophosphamide. The term“protein kinase inhibitor” refers to any substance that acts as aselective or non-selective inhibitor of a protein kinase. Examples ofspecific protein kinase inhibitors suitable for use in the VBIR forcancer include Lapatinib, AZD 2171, ET18OCH 3, Indirubin-3′-oxime,NSC-154020, PD 169316, Quercetin, Roscovitine, Triciribine, ZD 1839,5-Iodotubercidin, Adaphostin, Aloisine, Alsterpaullone, Aminogenistein,API-2, Apigenin, Arctigenin, ARRY-334543, Axitinib (AG-013736),AY-22989, AZD 2171, Bisindolylmaleimide IX, CCI-779, Chelerythrine,DMPQ, DRB, Edelfosine, ENMD-981693, Erbstatin analog, Erlotinib,Fasudil, Gefitinib (ZD1839), H-7, H-8, H-89, HA-100, HA-1004, HA-1077,HA-1100, Hydroxyfasudil, Kenpaullone, KN-62, KY12420, LFM-A13, Luteolin,LY294002, LY-294002, Mallotoxin, ML-9, MLN608, NSC-226080, NSC-231634,NSC-664704, NSC-680410, NU6102, Olomoucine, Oxindole I, PD 153035, PD98059, Phloridzin, Piceatannol, Picropodophyllin, PKI, PP1, PP2,PTK787/ZK222584, PTK787/ZK-222584, Purvalanol A, Rapamune, Rapamycin, Ro31-8220, Rottlerin, SB202190, SB203580, Sirolimus, SL327, SP600125,Staurosporine, STI-571, SU1498, SU4312, SU5416, SU5416 (Semaxanib),SU6656, SU6668, syk inhibitor, TBB, TCN, Tyrphostin AG 1024, TyrphostinAG 490, Tyrphostin AG 825, Tyrphostin AG 957, U0126, W-7, Wortmannin,Y-27632, Zactima (ZD6474), ZM 252868. gefitinib (Iressa®), sunitinibmalate (SUTENT; SU11248), erlotinib (TARCEVA: OSI-1774), lapatinib(GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib(PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec®; ST1571),dasatinib (BMS-354825), leflunomide (SU101), vandetanib (ZACTIMA;ZD6474), and nilotinib.

In some particular embodiments, the tyrosine kinase inhibitor issunitinib malate Sorafenib tosylate, or Axitinib. Sunitinib malate,which is marketed by Pfizer Inc. under the trade name SUTENT, isdescribed chemically as butanedioic acid, hydroxy-, (2S)-, compound withN-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-H-pyrrole-3-carboxamide(1:1). The compound, its synthesis, and particular polymorphs aredescribed in U.S. Pat. No. 6,573,293. Sunitinib malate has been approvedin the U.S. for the treatment of gastrointestinal stromal tumor,advanced renal cell carcinoma, and progressive, well-differentiatedpancreatic neuroendocrine tumors in patients with unresectable locallyadvanced or metastatic disease. The recommended dose of sunitinib malatefor gastrointestinal stromal tumor (GIST) and advanced renal cellcarcinoma (RCC) for humans is 50 mg taken orally once daily, on aschedule of 4 weeks on treatment followed by 2 weeks off (Schedule 4/2).The recommended dose of sunitinib malate for pancreatic neuroendocrinetumors is 37.5 mg taken orally once daily. In the VBIR for cancer,sunitinib malate may be administered orally in a single dose or multipledoses. Typically, sunitinib malate is delivered for two, three, four ormore consecutive weekly doses followed by an “off” period of about 1 or2 weeks, or more where no sunitinib malate is delivered. In oneembodiment, the doses are delivered for about 4 weeks, with 2 weeks off.The effective amount of sunitinib malate administered orally to a humanis typically below 40 mg per person per day, such as 37.5, 31.25, 25,18.75, 12.5, or 6.25 mg per person per day. In some embodiments,sunitinib malate is administered orally in the range of 1-25 mg perperson per day. In some other embodiments, sunitinib malate isadministered orally in the range of 6.25, 12.5, or 18.75 mg per personper dose. Other dosage regimens and variations are foreseeable, and willbe determined through physician guidance.

Sorafenib tosylate, which is marketed under the trade name NEXAVAR, hasthe chemical name is4-(4-{3-[4-Chloro-3-(trifluoromethyl)phenyl]ureido}phenoxy)-N-methylpyrid-ine-2-carboxamide.It is approved in the U.S. for the treatment of primary kidney cancer(advanced renal cell carcinoma) and advanced primary liver cancer(hepatocellular carcinoma). The recommended daily dose is 400 mg takenorally twice daily. In the VBIR for cancer provided by the presentdisclosure, the effective amount of sorafenib tosylate administeredorally is typically below 400 mg per person per day. In someembodiments, the effective amount of sorafenib tosylate administeredorally is in the range of 10-300 mg per person per day. In some otherembodiments, the effective amount of sorafenib tosylate administeredorally is between 10-200 mg per person per day, such as 10, 20, 60, 80,100, 120, 140, 160, 180, or 200 mg per person per day.

Axitinib, which is marketed under the trade name INLYTA, has thechemical name is(N-Methyl-2-[3-((E)-2-pyridin-2-yl-vinyl)-H-indazol-6-ylsulfanyl]-benzamide.It is approved for the treatment of advanced renal cell carcinoma afterfailure of one prior systemic therapy. The starting dose is 5 mg orallytwice daily. Dose adjustments can be made based on individual safety andtolerability. In the VBIR for cancer provided by the present disclosure,the effective amount of axitinib administered orally is typically below5 mg twice daily. In some other embodiments, the effective amount ofaxitinib administered orally is between 1-5 mg twice daily. In someother embodiments, the effective amount of axitinib administered orallyis between 1, 2, 3, 4, or 5 mg twice daily.

Examples of IEC enhancers that may be used in the VBIR for cancerprovided by the present disclosure include TNFR agonists, CTLA-4antagonists, TLR agonists, other PD-1 antagonists (such as BMS-936558and anti-PD-1 antibody CT-011), programmed cell death protein 1 ligand 1(PD-L1) antagonists (such as BMS-936559), lymphocyte-activation gene 3(LAG3) antagonists, and T cell Immunoglobulin- andmucin-domain-containing molecule-3 (TIM-3) antagonists. Examples of TNFRagonists include agonists of OX40, 4-1BB (such as BMS-663513), GITR(such as TRX518), and CD40. Examples of specific CD40 agonists aredescribed in details herein below.

In certain other embodiments, the additional immune modulator is ananti-CD40 agonist antibody. The antibody can be a human, humanized orpart-human chimeric anti-CD40 antibody. Examples of specific anti-CD40monoclonal antibodies include the G28-5, mAb89, EA-5 or S2C6 monoclonalantibody, and CP870893. In a particular embodiment, the anti-CD40agonist antibody is CP870893 or dacetuzumab (SGN-40).

CP-870,893 is a fully human agonistic CD40 monoclonal antibody that hasbeen investigated clinically as an anti-tumor therapy. The structure andpreparation of CP870,893 is disclosed in WO2003040170, in which antibodyCP870,893 is identified as antibody “21.4.1”. The amino acid sequencesof the heavy chain and light chain of CP-870,893 are set forth in SEQ IDNO: 46 and SEQ ID NO: 48, respectively, as well as in Table 7, inWO2003040170. In clinical trials, CP870,893 was administered byintravenous infusion at doses generally in the ranges of 0.05-0.25 mg/kgper infusion. In the VBIR for cancer provided by the present disclosure,CP-870,893 may be administered intradermally, subcutaneously, ortopically. The effective amount of CP870893 to be administered in theregimen is generally below 0.2 mg/kg, typically in the range of 0.01mg-0.15 mg/kg, or 0.05-0.1 mg/kg.

Dacetuzumab (also known as SGN-40 or huS2C6; CAS number 88-486-59-9) isanother anti-CD40 agonist antibody that has been investigated inclinical trials for indolent lymphomas, diffuse large B cell lymphomasand Multiple Myeloma. In the VBIR for cancer provided by the presentdisclosure, dacetuzumab may be administered intradermally,subcutaneously, or topically. The effective amount of dacetuzumab to beadministered is generally below 16 mg/kg, typically in the range of 0.2mg-14 mg/kg, or 0.5-8 mg/kg, or 1-5 mg/kg.

In still other embodiments, the additional immune modulator is an antiCTLA-4 antagonist. Examples of suitable anti-CTLA-4 antagonist includeanti-CTLA-4 antibodies (such as human anti-CTLA-4 antibodies, mouseanti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanizedanti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonalanti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, anti-CTLA-4domain antibodies), and inhibitors of CTLA-4 that agonize theco-stimulatory pathway. In some embodiments, the CTLA-4 inhibitor isIpilimumab or Tremelimumab.

Ipilimumab (marketed as YERVOY; also known as MEX-010, MDX-101, or byits CAS Registry No. 477202-00-9) is disclosed as antibody 10DI in PCTPublication No. WO 01/14424, incorporated herein by reference in itsentirety and for all purposes. Examples of pharmaceutical compositioncomprising Ipilimumab are provided in PCT Publication No. WO 2007/67959.Ipilimumab is approved in the U.S. for the treatment of unresectable ormetastatic melanoma. In the methods provided by the present invention,Ipilimumab may be administered intradermally or subcutaneously. Theeffective amount of Ipilimumab administered locally is typically in therange of 5-200 mg/dose per person. In some embodiments, the effectiveamount of Ipilimumab is in the range of 10-150 mg/dose per person perdose. In some particular embodiments, the effective amount of Ipilimumabis about 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.

Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonalantibody and has the CAS number 745013-59-6. Tremelimumab is disclosedas antibody 11.2.1 in U.S. Pat. No. 6,682,736, incorporated herein byreference in its entirety and for all purposes. In the VBIR for cancerprovided by the present invention, Tremelimumab may be administeredintravenously, intradermally, or subcutaneously. The effective amount ofTremelimumab administered intradermally or subcutaneously is typicallyin the range of 5-200 mg/dose per person. In some embodiments, theeffective amount of Tremelimumab is in the range of 10-150 mg/dose perperson per dose. In some particular embodiments, the effective amount ofTremelimumab is about 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg/doseper person.

In still other embodiments, the additional immune modulator is aToll-like Receptor (TLR) agonist. The term “toll-like receptor agonist”or “TLR agonist” refers to a compound that acts as an agonist of atoll-like receptor (TLR). This includes agonists of TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11 or a combinationthereof.

TLR agonists useful in the method of the present invention include bothsmall organic molecules and large biological molecules. Examples ofsmall molecule TLR agonists include 4-amino-alpha, alpha,2-trimethyl-IH-imidazo[4,5-c]qumolin-I-ethanol,N-(2-{2-[4-amino-2-(2-methoxyethyl)-IH-imidazo[4,5-c]quinolin-I-yl]ethoxy-}ethyl)-N-methylmorpholine-4-carboxamide,I˜(2˜amino-2-methylpropyl)-2-(ethoxymethyl-)-IH-imidazo[4,5-c]quinolin-4-amine,N-[4-(4-amino-2-ethyl-IH-imidazo[4,5-c]quinolin-1-yl)butyl]methanesulfonamide,N-[4-(4-amino-2-propyl-IH-imidazo[4,5-c]quinolin-I-yl)butyl]methanesulfonamide,and imiquimod. Some TLR agonists particularly useful in the methods orregimen provided by the present disclosure are discussed in reviewarticle: Folkert Steinhagen, et al.: TLR-based immune adjuvants. Vaccine29 (2011): 3341-3355. In some embodiments, the TLR agonists are TLR9agonists, particularly CpG oligonucleotides (or CpG.ODN). A CpGoligonucleotide is a short nucleic acid molecule containing a cytosinefollowed by a guanine linked by a phosphate bond in which the pyrimidinering of the cytosine is unmethylated. Examples of particular CpGoligonucleotides useful in the methods provided by the presentdisclosure include:

(CpG 7909; SEQ ID NO: 49) 5′ TCGTCGTTTTGTCGTTTTGTCGTT3′;(CpG 24555; SEQ ID NO: 50) 5′ TCGTCGTTTTTCGGTGCTTTT3′; and(CpG 10103; SEQ ID NO: 51) 5′ TCGTCGTTTTTCGGTCGTTTT3′.

CpG7909, a synthetic 24mer single stranded, has been extensivelyinvestigated for the treatment of cancer as a monotherapy and incombination with chemotherapeutic agents, as well as an adjuvant forvaccines against cancer and infectious diseases. In the methods providedby the present disclosure, CpG7909 may be administered by injection intothe muscle or any other suitable methods. For use with a nucleicacid-based vaccine, such as a DNA vaccine, a CpG may be co-formulatedwith the vaccine in a single formulation and administered byintramuscular injection coupled with electroporation. The effectiveamount of CpG7909 by intramuscular, intradermal, or subcutaneousadministration is typically in the range of 10 μg/dose-10 mg/dose. Insome embodiments, the effective amount of CpG7909 is in the range of0.05 mg-14 mg/dose. In some particular embodiments, the effective amountof CpG7909 is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 05 1 mg/dose. OtherCpG oligonucleotides, including CpG 24555 and CpG 10103, may beadministered in similar manner and dose levels.

In the VBIR for cancer, the anti-PD-1 antagonist, the vaccine, and theadditional immune modulators may be administered either simultaneouslyor sequentially. In some embodiments, a vaccine is administeredsequentially with respect to the anti-PD-1 antagonist antibody, butsimultaneously (e.g., in a mixture) with respect to one or moreadditional immune modulators. In cases where a nucleic acid vaccine isadministered in combination with a CpG, the vaccine and CpG may becontained in a single formulation and administered together by anysuitable method. In some embodiments, the nucleic acid vaccine and CpGin a co-formulation (mixture) is administered by intramuscular injectionin combination with electroporation.

Formulations

Therapeutic formulations of the anti-PD-1 antagonist antibody used inaccordance with the present invention are prepared for storage by mixingan antibody having the desired degree of purity with optionalpharmaceutically acceptable carriers, excipients or stabilizers(Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and maycomprise buffers such as phosphate, citrate, and other organic acids;salts such as sodium chloride; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens, such asmethyl or propyl paraben; catechol; resorcinol; cydohexanol; 3-pentanol;and m-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Liposomes containing the anti-PD-1 antagonist antibody are prepared bymethods known in the art, such as described in Epstein, et al., Proc.Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad.Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing(2000).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic anti-PD-1 antagonist antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from about 0.1 to about 500 mg of the active ingredient ofthe present invention. The tablets or pills of the novel composition canbe coated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can comprise fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing an anti-PD-1antagonist antibody with Intralipid™ or the components thereof (soybeanoil, egg phospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

Kits

The invention also provides kits comprising any or all of the antibodiesdescribed herein. Kits of the invention include one or more containerscomprising an anti-PD-1 antagonist antibody described herein andinstructions for use in accordance with any of the methods of theinvention described herein. Generally, these instructions comprise adescription of administration of the anti-PD-1 antagonist antibody forthe above described therapeutic treatments. In some embodiments, kitsare provided for producing a single-dose administration unit. In certainembodiments, the kit can contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments, kits containing single and multi-chambered pre-filledsyringes (e.g., liquid syringes and lyosyringes) are included.

In some embodiments, the antibody is a human antibody. In someembodiments, the antibody is a humanized antibody. In some embodiments,the antibody is a monoclonal antibody. The instructions relating to theuse of an anti-PD-1 antibody generally include information as to dosage,dosing schedule, and route of administration for the intended treatment.The containers may be unit doses, bulk packages (e.g., multi-dosepackages) or sub-unit doses. Instructions supplied in the kits of theinvention are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an anti-PD-1 antibody. The container may further comprisea second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Biological Deposit

Representative materials of the present invention were deposited in theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, USA, on Apr. 29, 2014. Vector msb7-LC having ATCCAccession No. PTA-121182 is a polynucleotide encoding the mAb7 lightchain variable region, and vector mab7-HC having ATCC Accession No.PTA-121183 is a polynucleotide encoding the mAb7 heavy chain variableregion. The deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Pfizer, Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

EXAMPLES Example 1 Effect of Anti-PD-1 Antibody on IFN-γ and TNFSecretion

This example illustrates the effect of anti-PD-1 antibody on IFN-γ andTNF secretion in a mixed lymphocyte reaction (MLR) assay.

Primary human T cell isolated from whole blood (Stanford Universityblood bank) were activated by allogeneic dendritic cells (DC) expressinghigh levels of PD-L1 and PD-L2, that were previously differentiatedusing IL-4 and GM-CSF from CD14+ myeloid cells. In this study, thefollowing antibodies were used: isotype control (IgG4 kappa hingestabilized), anti-PD-1 antogonist antibody C1, anti-PD-1 antagonistantibody C2, anti-PD-1 antagonist antibody C3, EH12.1 (BD Biosciencesmouse anti human anti-PD-1 antibody, mouse isotype IgG1 Kappa), mAb7-G4,mAb15-G4, mAb-AAA, mAb15-AAA (G4=IgG4 hinge stabilized; AAA=mutant IgG1which does not bind FcγR). Antibodies were tested at the followingconcentrations: 0, 0.1, 1, or 10 μg/ml).

For the MLR assay, cultures were incubated with test or control antibodyin 96 well plates in triplicates at ratios of 1:10 DC: T cells andincubated in humidified incubator at 37° C. with 5% CO₂. Supernatantswere harvested at day 5 and cytokines were measured using CytometricBead Array (CBA) using Human Soluble Protein Flex Set System kit (BDBiosciences, cat #558265) according to the manufacturer's protocol withthe following human analytes: IFNγ (BD Biosciences, cat #558269), TNF(BD Biosciences, cat #558273). Briefly, 96 well filter plates(Millipore, cat #MSBVN1250) were washed with Wash Buffer (BD Biosciencesproprietary formula) and aspirated by vacuum manifold. Standardsprovided by the kit and samples were diluted in Assay Diluent (BDBiosciences proprietary formula) and added to the plates with CaptureBeads (capture bead are beads coated with antibodies for a specificsoluble protein coated with a distinct fluorescence). Plates were mixedfor 5 minutes at 500 rpm using a plate shaker, and incubated for 1 hourat room temperature. Detection Reagent (phycoerythrin (PE)-conjugatedantibodies, provided by the k) was added to the plates and plates weremixed for 5 minutes. Plates were incubated for 2 hours at roomtemperature. Then were with wash Buffer for 5 minutes, and samples wereacquired on the BD Fortessa platforms. Data were analyzed using FCAPArray v3 (BD). The results of the MLR assay are shown in Tables 6A and Bbelow. Table 6A shows IFNγ levels (in pg/ml), and Table shows 6B TNFlevels (in pg/ml). Data are presented as an average±S.E.M of biologicaltriplicates. Samples are a representative of one MLR experiment.

TABLE 6A IFNγ secretion IFNγ Levels (pg/ml) Antibody Concentration(μg//ml) 0 0.1 1 10 Anti-body Ave SE Ave SE Ave SE Ave SE IsotypeControl 1190.598 173.4735 3760 367.4262 3693.972 1033.879 3525.655744.676 EH12.1 1190.598 173.4735 3443.495 749.905 6196.637 576.90227111.465 2619.065 mAb15-G4 1190.598 173.4735 4728.295 2.035 8893.595365.125 7790.95 1700.012 mAb7-G4 1190.598 173.4735 8567.203 2085.82611876.86 1259.788 11794.82 1827.243 mAb7-AAA 1190.598 173.4735 10978.781006.925 10177.68 1907.027 9048.097 2022.583 mAb15-AAA 1190.598 173.47359068.905 1332.045 7083.987 2109.455 8644.313 1797.077 C1 1190.598173.4735 5891.74 583.57 6992.53 338.89 7523.7 1907.073 C2 1190.598173.4735 3433.687 52.36606 8121.305 195.315 4295.95 3124.257 C3 1190.598173.4735 9698.06 529.47 11650.97 143.025 8282.573 2332.477

TABLE 6B TNF secretion TNF Levels (pg/ml) Antibody Concentration(μg//ml) 0 0.1 1 10 Anti-body Ave SE Ave SE Ave SE Ave SE IsotypeControl 371.3233 17.01197 407.4133 49.58195 486.7167 14.42418 501.175.033334 EH12.1 371.3233 17.01197 571.1233 60.43963 667.0033 35.72991799.3033 7.836955 mAb15-G4 371.3233 17.01197 743.1167 56.75547 686.3245.63348 798.8533 9.143657 mAb7-G4 371.3233 17.01197 730.47 33.35488793.05 21.19019 930.6233 16.04937 mAb7-AAA 371.3233 17.01197 795.113358.01784 773.32 32.32587 798.68 17.37746 mAb15-AAA 371.3233 17.01197803.87 34.20712 869.4867 43.04225 731.33 37.8211 C1 371.3233 17.01197641.5533 58.65366 809.1767 55.78437 656.6467 36.03128 C2 371.323317.01197 724.9133 35.53936 769.6367 22.06726 772.5367 49.44021 C3371.3233 17.01197 692.1067 39.12236 642.25 21.44117 744.0067 49.58307

Treatment of activated T cells with anti-PD-1 antagonist antibodiesresulted in increased IFNγ levels compared to isotype control (Table6A). For example, treatment with 0.1 μg/ml mAb15-G4 and mAb7-G4 resultedin an IFNγ level of 4728.295±2.035 pg/ml and 8567.203±2085.826 pg/ml,respectively. Treatment with 1 μg/ml mAb15-G4 and mAb7-G4 resulted in anIFNγ level of 8893.595±365.125 pg/ml and 11876.86±1259.788 pg/ml,respectively. Treatment with 10 μg/ml mAb15-G4 and mAb7-G4 resulted inan IFNγ level of 7790.95±1700.012 pg/ml and 11794.82±1827.243 pg/ml,respectively. In contrast, treatment with 0.1, 1, or 10 μg/ml isotypecontrol resulted in IFNγ levels of 3760±367.4262 pg/ml,3693.972±1033.879 pg/ml, and 3525.655±744.676 pg/ml, respectively.

Treatment of activated T cells with anti-PD-1 antagonist antibodiesresulted in increased TNF levels compared to isotype control (Table 6B).For example, treatment with 0.1 μg/ml mAb15-G4 or mAb7-G4 resulted in aTNF level of 743.1167±56.75547 pg/ml and 730.47±33.35488 pg/ml,respectively. Treatment with 1 μg/ml mAb15-G4 and mAb7-G4 resulted in aTNF level of 686.32±45.63348 pg/ml and 793.05±21.19019 pg/ml,respectively. Treatment with 10 μg/ml mAb15-G4 or mAb7-G4 resulted in aTNF level of 798.853±9.14366 pg/ml and 930.623±16.0494 pg/ml,respectively. In contrast, treatment with 0.1, 1, or 10 μg/ml isotypecontrol resulted in TNF levels of 407.4133±49.58195 pg/ml,486.7167±4.4241 pg/ml, and 501.17±5.033334 pg/ml, respectively.

These results demonstrate that anti-PD-1 antibodies mAb7 and mAb15stimulate IFNγ and TNF secretion from T cells at least as well as orbetter than anti-PD-1 antibodies C1, C2, and C3.

A second MLR study was conducted to test the effect of lower antibodyconcentrations on T cell activation. Primary human T cell isolated fromwhole blood were activated as described above. The following antibodieswere tested in the second study: mAb7 (G4), mAb15 (G4), C1 (G4), EH12.1,and G4 isotype control. Antibodies were tested at the followingconcentrations: 0.0001, 0.001, 0.01, 0.1, 1, and 10 pig/ml. The MLRassay was conducted as described above. Results are summarized in Tables7A and 7B below.

TABLE 7A IFNγ secretion IFNγ Levels (pg/ml) Antibody Concentration(μg/ml) Anti-body 0.0001 0.001 0.01 0.1 1 10 Isotype Control  558.9629 ± 753.3767 ± 1074.37 ± 1667.96 ±  1867.96 ±  2501.293 ±  489.4828 291.6092  324.2031  144.7286  282.7461  220.1829 EH12.1 1703.907 ±2284.153 ± 4384.477 ± 4726.87 ±  9914.05 ± 15110.19 ±  417.5669 408.1215  396.1451 1201.688  1188.064  1864.176 C1 1774.38 ± 2804.017 ±4148.123 ± 6883.113 ±  9598.413 ± 10283.24 ±  290.6059  598.5177 194.2466 1480.168  617.4762  1008.533 mAb7 (G4) 2082.07 ± 3062.09 ±5067.823 ± 7082.667 ± 11928.81 ± 11862.13 ±  720.9931  370.2791 111.4903 1336.082  1457.723  800.586 mAb15 (G4) 1678.27 ± 1410.758 ±4734.49 ± 5416 ±  9140.337 ± 10992.13 ±  233.82  439.9474  322.20871054.075  1320.499  1008.533

TABLE 7B TNF secretion TNF Levels (pg/ml) Antibody Concentration (μg/ml)Anti-body 0.0001 0.001 0.01 0.1 1 10 Isotype Control 452.7133 ± 282.9287± 310.9144 ±  358.948 ±  338.1107 ±  331.008 ±  62.85  56.77266  21.7811 81.09122  46.88385  31.35559 EH12.1 752.87 ± 687.3733 ± 859.0367 ± 744.98 ±  978.7567 ± 1950.73 ±  32.81303  29.09567  89.3586  131.3658 227.9979  155.2098 C1 446.98 ± 444.7867 ± 465.0933 ± 1045.45 ± 997.6067 ±  895.7267 ±  26.89211  59.03216  65.75044  146.9018 79.17846  60.92022 mAb7 (G4) 227.6 ± 394.4233 ± 452.65 ± 1089.377 ±1583.52 ± 1419.88 ±  50.63436  30.47005  30.64335  174.7824  267.2131 108.711 mAb15 (G4) 494.7967 ± 489.2333 ± 593.34 ±  811.16 ± 1143.54 ±1109.063 ±  48.18105  30.63302  65.87622  89.50238  136.3954  57.70232

Treatment of activated T cells with anti-PD-1 antagonist antibodiesresulted in increased IFNγ levels compared to isotype control (Table7A). In cultures without antibody, the IFNγ level was 901.453±216.472pg/ml. In cultures given 0.0001, 0.001, 0.01, 0.1, 1, or 10 μg/mlisotype control antibody, the IFNγ levels were 558.9629±489.4828 pg/ml,753.3767±291.6092 pg/ml, 1074.37±324.2031 pg/ml, 1667.96±144.7286 pg/ml,1867.96±282.7461 pg/ml, 2501.293±220.1829 pg/ml, respectively. Incontrast, in cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10μg/ml mAb7 (G4), the IFNγ levels were 2082.07±720.9931 pg/ml,3062.09±370.2791 pg/ml, 5067.823±111.4903 pg/ml, 7082.667±1336.082pg/ml, 11928.81±1457.723 pg/ml, 11862.13±800.586 pg/ml, respectively. Incultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10 μg/ml mAb15(G4), the IFNγ levels were 1678.27±233.82 pg/ml, 1410.758±439.9474pg/ml, 4734.49±322.2087 pg/ml, 5416±1054.075 pg/ml, 9140.337±1320.499pg/ml, and 10992.13±1008.533 pg/ml, respectively.

Treatment of activated T cells with anti-PD-1 antagonist antibodiesresulted in increased TNF levels compared to isotype control (Table 7B).In cultures without antibody, the TNF level was 365.523±84.6607 pg/ml.In cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10 μg/mlisotype control antibody, the TNF levels were 452.7133±62.85 pg/ml,282.9287±56.77266 pg/ml, 310.9144±21.7811 pg/ml, 358.948±81.09122 pg/ml,338.1107±46.88385 pg/ml, and 331.008±31.35559 pg/ml, respectively. Incontrast, in cultures treated with 0.0001, 0.001, 0.01, 0.1, 1, or 10μg/ml mAb7 (G4), the TNF levels were 227.6±50.63436 pg/ml,394.4233±30.47005, 452.65±30.64335 pg/ml, 1089.377±174.7824 pg/ml,1583.52±267.2131 pg/ml, and 1419.88±108.711 pg/ml, respectively. Incultures given 0.0001, 0.001, 0.01, 0.1, 1, or 10 μg/ml mAb15 (G4), theTNF levels were 494.7967±48.1810 pg/ml, 489.2333±30.63302 pg/ml,593.34±65.87622 pg/ml, 811.16±89.50238 pg/ml, 1143.54±136.3954 pg/ml,and 1109.063±57.70232 pg/ml, respectively.

These results demonstrate that that anti-PD-1 antibodies mAb7 and mAb15block PD-1 signaling and promote IFNγ and TNF secretion from primaryhuman T cells.

Example 2 Effect of Anti-PD-1 Antibodies on T Cell Proliferation

This example illustrates the effect of anti-PD-1 antibodies on T cellproliferation.

In this study, T cell proliferation was measured in an MLR assay inwhich T cells were cultured in the presence of anti-PD-1 antagonist orisotype control antibodies.

For the MLR, primary human T cell isolate from whole blood (obtainedfrom Stanford University blood bank) were activated by allogeneicdendritic cells (DC) expressing high levels of PD-L1 and PD-L2, thatwere previously differentiated using IL-4 and GM-CSF from CD14+ myeloidcells. Two experiments were conducted.

In the first experiment, mAb7 (IgG4 kappa hinge stabilized), mAb15 (IgG4kappa hinge stabilized), C1, EH12.1 and isotype control were compared.In the second experiment, clones mAb7, mAb15, C2, EH12.1 and isotypecontrol were compared. In both experiments antibodies were added at thefollowing concentrations: 0, 0.0001; 0.001; 0.01; 0.1; 1 and 10 μg/ml.

For both experiments, cultures were incubated with antibody in 96 wellplates in triplicates at ratios of 1:10 DC:T cells and incubated inhumidified incubator at 37° C. with 5% CO₂. On day 5, cultures werepulsed for 18 h with 1 μCi per well of [³H]-thymidine before harvesting.Plates then harvested on DNA specific filter papers (Perkin Elmer) usingHarvester96 (Tomtec Life Sciences). The radiolabeled filters werecovered with beta scintillation liquid (Perkin Elmer) and read inMicrobeta® counter plates (Perkin Elmer). Thymidine incorporation wereanalyzed as counts per minute (CPM). Results are shown as mean oftriplicates±SEM.

TABLE 8A Antibody Thymidine Incorporation (CPM) concentration isotype(μg/ml) control EH12.1 mAb7 mAb15 C1 0 183419.3 ± 183419.3 ± 183419.3 ±183419.3 ± 183419.3 ±  4049.932  4049.932  4049.932  4049.932  4049.9320.0001 205190 ± 227412.3 ± 226145 ± 217278.7 ± 211881.3 ±  7769.199 7769.199  9610.045  24472.76  10119.4 0.001 197943 ± 218722 ± 235367.3± 193192 ± 212198.3 ±  13904.69  13904.69  15199.75  11885.7  36451.790.01 175973.3 ± 253913.3 ± 265654.3 ± 219167.3 ± 232600 ±  10177.52 10177.52  12087.89  8928.691  26403.78 0.1 192495.3 ± 248270.7 ±264057.7 ± 257924 ± 278997.3 ±  16825.35  16825.35  10339.94  3376.669 18441.4 1 210104.7 ± 210104.7 ± 298696.7 ± 276531 ± 276141.3 ± 11484.23  11484.23  6164.177  3855.779  18516.97 10 206281.7 ± 294602.7± 301293 ± 286260.7 ± 264223.7 ±  16001.33  16001.33  6313.417  11483.22 9809.792

TABLE 8B Antibody Thymidine Incorporatin (CPM) concentration isotype(μg/ml) control EH12.1 mAb7 mAb15 C2 0 229959 ± 229959 ± 229959 ± 229959± 229959 ±  5794.112  27771.7  27771.7  5794.112  27771.7 0.0001 299107± 258428.3 ± 280415.3 ± 241112.7 ± 258069.7 ±  3193  36794.25  14101.94 40486.56  23962.19 0.001 277197 ± 272289 ± 260183 ± 233184 ± 267117.7 ± 17518  29320.04  24634.25  28899.06  16388.83 0.01 278072.3 ± 324891.3± 365625.3 ± 317377 ± 381936.3 ±  32671.62  3396.229  30171.07  31915.29 31901.57 0.1 268939.7 ± 342131.3 ± 380054.3 ± 360226.3 ± 381110.7 ± 12332.06  19839.88  9774.328  1802.69  16996.97 1 241164 ± 388757.7 ±392256.7 ± 421229 ± 401219 ±  13776.81  15684.37  15341.19  27865.13 1816.754 10 231897.7 ± 408098 ± 372889.7 ± 323441.3 ± 391925.3 ± 25865.95  20237.34  14826.49  64476.55  46054.82

Treatment of activated T cells with anti-PD-1 antagonist antibodies at aconcentration of 0.01 μg/ml or greater resulted in significantlyincreased T cell proliferation compared to isotype control (Tables 8Aand 8B).

For example, treatment with 0.01, 0.1, 1, or 10 μg/ml mAb7 resulted inthymidine incorporation rates of 365625.3±30171.07 CPM,380054.3±9774.328 CPM, 392256.7±15341.19 CPM, and 372889.7±14826.49 CPM,respectively (Table 8B). Treatment with 0.01, 0.1, 1, or 10 μg/mlmAb15-G4 and mAb7 resulted in thymidine incorporation rates of317377±31915.29 CPM, 360226.3±1802.69 CPM, 421229±27865.13 CPM, and323441.3±64476.55 CPM, respectively (Table 8B). In contrast, treatmentwith 0.01, 0.1, 1, or 10 μg/ml isotype control resulted in thymidineincorporation rates of 278072.3±32671.62 CPM, 268939.7±12332.06 CPM,241164±13776.81 CPM, and 231897.7±25865.95 CPM, respectively (Table 8B).

These results demonstrate that that anti-PD-1 antibodies mAb7 and mAb15block PD-1 signaling and promote proliferation of primary human T cells.

Example 3 Effect of Anti-PD-1 Antibodies in a Mouse Model of GvHD

This example illustrates the effect of anti-PD-1 antibodies on T cellproliferation and body weight loss in a mouse model of graft versus hostdisease (GvHD).

NOD-scid-IL-2 receptor gamma chain null (NSG) mice were used in thisstudy to test the effects of anti-PD-1 antagonist antibodies on T cellproliferation in vivo. Because NSG mice lack T cells and B cells andhave impaired NK cells, high engraftment of human cells is readilyachieved. When human PBMCs are engrafted in these mice, human T cellproliferation occurs and induces GvHD. The GvHD involves the myeloidcompartment of the host as well as the human cells. Massiveproliferation of human lymphocytes can be seen in the blood at earlystages followed by high infiltration of these cells into the mouseorgans, such as the liver, spleen, kidney, gut etc., resulting in bodyweight loss of the mice as well as skin lesions, hunched back, anddeath. The severity of the models depends on the donor PBMCs, and maydiffer between donors.

In this study, the following anti-PD-1 antagonist antibodies were used:mAb7 (human IgG4 hinge stabilized or AAA), mAb15 (human IgG4 hingestabilized), C1, C2, and C3. For the negative control, an isotypecontrol human IgG4 hinge stabilized antibody was used. Primary humanPBMCs were isolated from whole blood (Stanford University blood bank),using Ficoll gradient. 10⁷ human PBMCs were injected into NSG mice(females 8 weeks old, Jackson Laboratories). At day 0 mice wererandomized based on body weight and PBMCs were injected intravenously.For experiments 1-4, on day 2 and day 8 antibodies were administeredintraperitoneally at 10 mg/kg. For experiment 5, on day 2 and day 8,antibodies were administered intraperitoneally at 1 mg/kg or 10 mg/kg.Table 9 summarizes the antibodies used in each experiment.

TABLE 9 Antibodies Used in Experiments 1-5 Experiment ExperimentExperiment Experiment Experiment 1 2 3 4 5 isotype isotype isotypeisotype isotype control control control control control mAb7 mAb15 mAb15mAb15 mAb7 mAb15 mAb7 mAb7 mAb7 mAb7-AAA C1 C3 C2 C1

Bodyweight was measured periodically. Results are summarized in FIGS.1A-1E. Mice were bled periodically to assess T cell proliferation.Treatment with anti-PD-1 antibody accelerated disease course as measuredby rate of body weight loss. Compared to control mice, mice treated withanti-PD-1 antagonist antibody had more rapid body weight loss (FIGS.1A-1E).

Proliferation of human T cells was measured by flow cytometry using CD45as a marker (clone H130; BD Biosciences). Flow cytometry results aresummarized below in Table 10. T cell proliferation was higher in micetreated with anti-PD-1 antagonist antibody than in mice treated withisotype control. Higher percentage CD45 indicates higher level ofproliferation of the CD45 cells and therefore more severe GvHD.

In Experiment 1, the percentage of CD45 positive blood cells was 63.86%in control mice (Table 10). In contrast, the percentage of CD45 positiveblood cells in mice treated with anti-PD-1 antibody mAb7, mAb15, C1, ormAb7-AA was 80.34%, 77.62%, 77.26% and 76.9%, respectively (Table 10).

TABLE 10 T cell proliferation as measured by presence of CD45, animalstreated Experiment 1: % CD45 positive cells in blood at day 17Antibodies (10 mg/ml) control mAb7 mAb15 C1 mAb7-AAA 51.5 72.4 70 87.666.6 52 76.8 69 82 81 74.7 82.5 85.8 64.7 71 65.3 84 93.3 80 85.883 75.886 70 72 80 Average 63.86 80.34 77.62 77.26 76.8966 SEM 5.8926437192.80258809 5.608074536 4.48826247 3.937116896 Experiment 2: % CD45positive cells in blood at day 12 Antibodies control mAb7 C1 mAb15 59 8180 78.1 51 80.078 75 79.3 66 82.5 78 69.9 75 84 80 81.8 66 86 86 83.9Average 63.4 82.7156 79.8 78.6 SEM 4.480513 1.182785 2.012461 2.678152Experiment 3: % CD45 positive cells in blood at day 12 Antibodies (10mg/ml) control mAb7 C3 mAb15 51.5 72.4 70 72 66 80 69 74.8 74.7 82.585.8 86.9 65.3 84 93.3 90.2 75.8 86 70 79.4 Average 66.66 80.98 77.6280.66 SEM 4.875269 2.63638 5.608075 3.880013 Experiment 4: % CD45positive cells in blood at day 12 Antibodies (10 mg/ml) control mAb7 C2mAb15 67 92.23 83.1 88.1 77.98 88.71 82.3 74.8 78 82.5 85.8 86.9 90 9393.3 90.2 86.8 80.1 90 79.4 Average 79.956 87.308 86.9 83.88 SEM4.495211 2.890314 2.336932 3.253729 Experiment 5: % CD45 positive cellsin blood at day 10 Antibodies mAb7 mAb7 Control (1 mg/kg) (10 mg/kg) 7072.4 77 66 79 77 74.7 82.5 85.8 65.3 84 93.3 70 86 90 Average 69.2 80.7884.62 SEM 1.887127 2.668895 3.723305

In summary, mice treated with anti-PD-1 antibody had more rapid bodyweight loss and increased T cell proliferation compared to mice treatedwith isotype control. These results demonstrate that treatment withanti-PD-1 antibody stimulates proliferation of human T cells in vivo.

Example 4 Binding of Anti-PD-1 Antibodies

This example illustrates anti-PD-1 antibody binding on activated human Tcells and cynomolgus monkey (cyno) T cells.

Primary human T cells were isolated from PBMCs (Stanford Universityblood bank) using a human PAN T cell isolation kit according to themanufacturer protocol (Miltenyi Biotec; 130-096-353). Cyno PBMCs werepurchased from (BioreclamationIVT), and PAN T cells were isolated usingnon-human primate PAN T cell isolation kit according to the manufacturerprotocol (Miltenyi Biotec; 130-091-993). Human T cells were activatedfor 3 days with DYNABEADS™ human T-Activator CD3/CD28 for cell expansionand activation (Life Technologies; 11131D). Ratio of beads to cell usedwas 1:1 bead:T cell, respectively. Cyno T cells were activated for 3days using T Cell Activation/Expansion Kit, non-human primate accordingto the manufacturer protocol (Miltenyi Biotec; 130-092-919). Ratio ofbeads to cells used was 1:1 bead: T cell; respectively. After 3 dayscultures were harvested beads were separated form activated T cell usingmagnetic force. Cells were washed and incubated with FACS buffer(including 2% FBS) and human Fc Receptor binding inhibito (AffymetrixeBioscience cat. no. 16-9161-73). For cyno cells were used Fc Blockreagent (BD Biosciences cat. no. 564765). Cells were incubated for 10minutes at room temperature and then were then stained with live deadcolor to exclude dead cells (LIVE/DEAD® Fixable Blue Dead Cell StainKit, for UV excitation; catalog #A10346) for another 5 minutes. AntiPD-1 antibodies were added (concentrations of anti-PD-1 clones wereincubated on cell in 1:3 serial dilution ratios starting 10 μg/ml-0μg/ml) 1×10 cells were used in each reaction in total of 100 μl andcells were incubated on ice for 30 minutes. Cells then washed with FACSbuffer to remove access of primary antibodies and incubated with antihuman (AffiniPure F(ab′)₂ Fragment Donkey Anti-Human IgG (H+L) secondaryconjugated with Allophycocyanin (APC); cat. no. 709-136-149). Cells werestained for 30 minutes on ice. Cells were washed and kept on ice untilread using BD LSRFortessa Cell Analyzer, (BD Biosciences, cat. no.647465). Data were analyzed using FlowJo™ software. Results aresummarized in FIGS. 2A and 2B.

FIG. 2A shows EC50 measured for anti-PD-1 antibody binding to humanactivated cells, and FIG. 2B shows EC50 measured for anti-PD-1 antibodybinding to cyno activated cells. Anti-PD-1 antibodies mAb7 and C1 bindactivated T cells with similar EC50 (FIGS. 2A and 2C).

Example 5 Inhibition of PD-L1 Binding by Anti-PD-1 Antibody

This example illustrates inhibition of PD-1 ligand (PD-L1) binding byanti-PD-1 antibody.

Primary human T cells were isolated from PBMCs (Stanford Universityblood bank) using human PAN T cell isolation kit according to themanufacturer protocol (Miltenyi Biotec; 130-096-353). Cynomolgus MonkeyPBMCs were purchased from (BioreclamationIVT), and PAN T cells wereisolated using non-human primate PAN T cell isolation kit according tothe manufacturer protocol (Miltenyi Biotec; 130-091-993). Human T cellswere activated for 3 days with DYNABEADS™ human T-Activator CD3/CD28(for cell expansion and activation, Life Technologies; 11131D). Ratio ofbeads to cell used was 1:1; respectively. Cyno T cells were activatedfor 3 days using T Cell Activation/Expansion Kit, non-human primateaccording to the manufacturer protocol (Miltenyi Biotec; 130-092-919).Ratio of beads to cells used was 1:1; respectively. After 3 dayscultures were harvested beads were separated form activated T cell usingmagnetic force. Cells were washed and incubated with FACS buffer(including 2% FBS) and human Fc Receptor binding inhibitor (AffymetrixeBioscience; cat. no. 16-9161-73). For cyno cells were used Fc Blockreagent (BD Biosciences; cat. no. 564765). Cells were incubated for 10minutes at room temperature and then were stained with live dead colorto exclude dead cells (LIVE/DEAD® Fixable Blue Dead Cell Stain Kit, forUV excitation; cat. no. A10346) for another 5 minutes. Human RecombinantPD-L1 Fc (R&D Systems, cat. no. 156-B7) or buffer alone was incubatedwith cells at 10 ng/ml. Each ligand was incubated separately andincubated on ice for 30 minutes. Cells then were washed and incubatedwith anti PD-1 antibodies (concentrations of anti-PD-1 clones wereincubated on cell in 1:3 serial dilution ratios starting at 1 μg/ml-0μg/ml) 1×10⁶ cells were used in each reaction in total of 100 μl andcells were incubated on ice for 30 minutes. Cells then washed with FACSbuffer to remove access of primary antibodies and incubated withanti-human kappa conjugated with Allophycocyanin (APC) (LifeTechnologies; cat no. MH10515). Cells were stained for 30 minutes onice, then washed and kept on ice until read using BD LSRFortessa CellAnalyzer, (BD Biosciences, cat. no. 647465). Data were analyzed usingFlowJo™ software and Mean fluoresce intensity (MFI) and geometricalmeans (Geo.M) of APC staining on live cells were calculated in theFlowJo™ software. After Geo mean calculation IC50 were calculated usingin GraphPD Prism software. Results are summarized in Tables 11 and 12below.

TABLE 11 Anti-PD-1 blockade of PD-L1 binding to PD-1 on human T cellsAntibody concentration Geo Mean (μg/ml) mAb7 C1 0.0083375 107 1430.00416875 129 190 0.002084375 162 245 0.001042188 205 327 0.000521094482 415 0.000260547 358 469 0.000130273 445 484 0.000065137 503 4580.000032568 450 420 IC50 (μM) 0.001117 0.00224

TABLE 12 Anti-PD-1 blockade of PD-L1 binding to PD-1 on cyno T cellsAntibody concentration Geo Mean (μg/ml) mAb7 C1 0.0083375 114 1080.00416875 135 144 0.002084375 174 183 0.001042188 240 264 0.000521094322 325 0.000260547 440 404 0.000130273 494 469 0.000065137 491 4720.000032568 410 406 IC50 (μM) 0.00092 0.00108

These results demonstrate that anti-PD-1 antibodies mAb7 and C1 inhibitPD-L1 binding to human and cyno T cells with similar IC50.

Example 6 Effect of Anti-PD-1 Antibody on T Cell Proliferation

This example illustrates the effect of anti-PD-1 antibody on T cellproliferation. CD4 and CD8 (AllCells, LLC) were activated for 2 dayswith DYNABEADS™ human T-Activator CD3/CD28 (or cell expansion andactivation (Life Technologies; 11131D). Ratio of beads to cell used was1:1; respectively to induce PD-1. At day 2, cultures were harvested andbeads were separated from activated T cell using magnetic force. Cellsthen activated on PD-L1 expressing dendritic cells and cells wereincubated with different anti-PD-1 clones at 1 μg/ml in 96 well platesin triplicates at ratios of 1:10 DC: T cells and incubated in humidifiedincubator at 37° C. with 5% CO₂. On day 3, cultures were pulsed for 18 hwith 1 μCi per well of [³H]-thymidine before harvesting. Plates thenharvested on DNA specific filter papers (PerkinElmer) using Harvester96(Tomtec Life Sciences). The radiolabeled filters were covered with betascintillation liquid (Perkin Elmer) and read in Microbeta® counterplates (Perkin Elmer). Thymidine Incorporation were analyzed as countsper minute (CPM). Results are shown as mean of triplicates±SEM in FIGS.3 and 4.

Example 7 Kinetic and Affinity Determination of Human, Cynomolgus Monkeyand Mouse PD-1 Interacting with Humanized Anti-PD-1 Antibodies

This example illustrates binding of anti-PD-1 antibodies to human, cyno,or mouse PD-1.

All interaction analysis was performed on label-free biosensors at 25°C. unless stated otherwise. Surface plasmon resonance biosensors(ProteOn-XPR™ from BioRad™, and Biacore 2000™ and Biacore T200™ from GELife Sciences) were used to study human and cynomolgus monkey PD-1 and abiolayer interferometry biosensor (Octet-Red384, Fortebio/Pall LifeSciences) was used to study mouse PD-1. ProteOn experiments wereperformed in PBS pH 7.4+0.01% Tween-20 (PBST) running buffer. Biacoreexperiments were performed in 10 mM Hepes pH 7.4, 150 mM NaCl, 0.05%Tween-20 (HBST+) and Octet experiments were performed in HBST+ with 1g/l BSA. The ProteOn data were processed in the ProteOn Managersoftware, the Biacore data were processed in Biaevaluation, and theOctet data were simply aligned to zero in the control software. The SPRdata were double-referenced (Myszka, 1999, J Mol Recognit 12(5):279-284)and fit globally to a simple Langmuir model to determine the equilibriumdissociation constant, K_(D), from the ratio of the kinetic rateconstants (K_(D)=k_(d)/k_(a)).

Calibration-Free Concentration Analysis (CFCA)

The active concentration of human PD-1 (hPD-1) monomer (SinoBiologicals, cat. no. 10377-H08H) for use as analyte in the kineticexperiments with immobilized IgGs was determined empirically using aCFCA assay on a Biacore T200™ equipped with CM5 sensor chip. To preparethe surfaces for these experiments, a high capacity (approximately12,000 RU) of mAb15 hlgG4 (or in some experiments, competitor antibodyC2-hlgG1) was amine-coupled onto flow cell 2, leaving flow cell 1 blank(just “activated and blocked”, without any IgG) to provide a referencesurface. The hPD-1 samples were injected at nominal concentrations of0.1, 1, and 10 μg/ml for 36 sec at both low (5 μl/min) and high (100μl/min) flow rates. Surfaces were regenerated with a cocktail of 2:1 v/vPierce IgG elution buffer (pH 2.8):4 M NaCl. Data were analyzed in theCFCA tool in the T200 software to derive an apparent activity value forthe hPD-1 analyte, which was used to correct its “nominal” proteinconcentration, as determined by absorbance at 280 nm with appropriateextinction coefficient, into an “active” protein concentration. Somelots were found to be 32% active, while others were 100% active.

Kinetic Analysis of Human PD-1 (hPD-1) Binding to Amine-Coupled mAb7,mAb15, C1, C2, C3, and C4, mAbs

A ProteOn-XPR36 equipped with GLC sensor chips (BioRad™, Hercules,Calif.), was used to determine the kinetics and affinity of hPD-1monomer binding to a panel of amine-coupled anti-hPD-1 mAbs (mAb7,mAb15, C1, C2, C3, and C4) in PBST running buffer. The surfaces forthese experiments were prepared in three steps; (1) the ligand channelswere minimally activated for two min using a freshly prepared mixture ofthe activation reagents at final 0.8 mM EDC and 0.2 mM sulfo-NHS inwater, (2) the IgGs were coupled for three min at 15 μg/ml in 10 mMsodium acetate pH 4.5, and (3) excess reactive esters were blocked forthree min with 1 M ethanolamine HCl pH 8.5. Final levels of coupled IgGranged from 400 RU to 1157 RU. The hPD-1 monomer was injected in aone-shot kinetic mode (Bravman et al., 2006, Anal Biochem358(2):281-288) along the “analyte” channels as a threefold dilutionseries with top “active” concentrations of 30, 44 or 36 nM, depending onthe experiment. Association and dissociation times were 3 min and 20min, respectively and all analytes were injected in duplicate bindingcycles. Surfaces were regenerated with a cocktail of 2:1 v/v Pierce IgGelution buffer (pH 2.8):4 M NaCl.

TABLE 13 Kinetic analysis of hPD-1 monomer binding to amine-coupled IgGsk_(a) k_(d) K_(D) (pM) IgG (1/Ms) × 10⁵ (1/s) × 10⁻⁴ at 25° C. mAb7 IgG1AAA 4.97 <0.43* <86 mAb7 IgG4 4.53, 4.45 <0.43* <94, <96 (N = 2) mAb15IgG1 AAA 8.13 <0.43* <53 mAb15 IgG4 7.32 <0.43* <58 C4 hIgG1 5.57, 4.845.25, 5.89  943, 1217 (N = 2) C3 hIgG2 4.56 5.21 1143 C3 hIgG4 5.59 4.03721 C2 hIgG2 4.88 6.07 1244 C2 hIgG4 5.84 4.33 741 C1 hIgG2 14.8 7.55510 C1 11.1, 8.34 5.57, 5.73 502, 687 (N = 2)The interactions of hPD-1 monomer with mAb7 and mAb15 showed no visibledecay in binding response within the allowed dissociation phase, so anupper limit was placed on their k_(d) and K_(D) values, according to the“5% rule” (Katsamba et al, 2008, Anal Biochem 352(2):208-221) wasapplied to place an upper limit on their k_(d) and K_(D) values. N=2refers to two independent experiments on different chips.Cross-Reaction of mAb7 and mAb15 to Cynomolgus Monkey PD-1

The binding kinetics of recombinant purified Fab fragments (mAb7 andmAb15) to both hPD-1-hFc1 (R&D systems cat. no. 1086PD) andcynoPD-1-hFc1 (prepared in-house) was determined using a Biacore 2000™equipped with a CM4 sensor chip and HBST+ running buffer. An anti-hFcpolyclonal antibody was amine-coupled to the chip and used to captureapproximately 90 RU hPD-1-hFc1 and 125 RU cynoPD-1-hFc1 on flow cells 2and 3, leaving flow cell 1 blank (naked anti-hFc capture surface) toprovide a reference channel. Recombinant purified Fabs were injected fortwo min as analyte at 0, 10, and 100 nM over freshly captured PD-1-hFc1fusion proteins, allowing a 15-min dissociation time. Capture surfaceswere regenerated using 75 mM phosphoric acid and the mAb7 Fab sampleswere injected in duplicate binding cycles. All Fab/PD-1 complexes werevery stable, such that none of the interactions showed any visible decayin their binding responses within the allowed dissociation time, so the“5% rule” (Katsamba et al, 2008) was applied to place an upper limit ontheir k_(d) and K_(D) values.

TABLE 14 Affinity determination of mAb7 and mAb15 Fabs towardshPD-1-hFc1 and cynoPD-1-hFc1 fusion proteins k_(a) *k_(d) K_(D) (pM)Analyte on chip (1/Ms) × 10⁵ (1/s) × 10⁻⁵ at 25° C. mAb7 Fab hPD-1-hFc15.67 <5.7 <101 mAb7 Fab cynoPD-1-hFc1 5.26 <5.7 <108 mAb15 FabhPD-1-hFc1 9.16 <5.7 <62 mAb15 Fab cynoPD-1-hFc1 8.24 <5.7 <69Temperature Dependence of the hPD-1 Binding Affinity Towards mAb15,mAb7, and C3

A Biacore T200™ equipped with CM4 sensor chip was used to determine thekinetics and affinities of hPD-1 monomer binding to a panel of hlgG4molecules (mAb15, mAb7, and C3) that were captured at low levels viaamine-coupled anti-hFc polyclonal antibody. The hlgG4 mAbs were capturedat 10 μg/ml on individual flow cells, leaving flow cell 1 blank to serveas a reference surface (naked capture surface). The hPD-1 was injectedat active concentrations of 0, 10, and 100 nM for three minutes allowingan 18-min dissociation phase. The capture surfaces were regenerated with75 mM phosphoric acid after each binding cycle

TABLE 15 Kinetic analysis of hPD-1 monomer binding as analyte toanti-hFc-captured hIgG4 molecules hIgG4 on chip Temp (25° C.) ka (1/Ms)kd (1/s) K_(D) (nM) mAb15 25 3.49 × 10⁵ 1.71 × 10⁻⁴ 0.49 mAb15 37 6.94 ×10⁵ 3.73 × 10⁻⁴ 0.54 mAb7  25 2.37 × 10⁵ 1.73 × 10⁻⁴ 0.73 mAb7  37 4.28× 10⁵ 3.63 × 10⁻⁴ 0.85 C3 25 2.14 × 10⁵ 2.25 × 10⁻³ 10.5 C3 37 9.70 ×10⁵ 1.56 × 10⁻² 16.1Cross-Reaction of mAb7 to Mouse-PD-1

An Octet-Red384 equipped with streptavidin sensor tips was used todetermine whether mouse PD-1 binds to mAb7. An avidity-prone assayformat was chosen to increase the detection sensitivity of the assay.Sensors were coated with biotinylated anti-human kappa polyclonal andused to capture a panel of hlgG4 anti-hPD-1 mAbs (mAb7, C1, C2, and C3)at 10 μg/ml; each mAb was captured on eight sensors. As a positivecontrol, eight streptavidin sensors were coated with biotinylated J43(eBioSciences), an anti-mouse PD-1 antibody. Each mAb-coated sensor wasexposed to the following analytes; buffer, 1 μM binding sites mousePD-1-hFc, 1 μM binding sites hPD-1-hFc (positive control) or 1 μMbinding sites hGHR-hFc (negative control). All recombinant Fc-fusionproteins were from R&D systems. Each analyte/mAb interaction wastherefore tested on duplicate sensors.

Anti-PD-1 antibodies C1, C2, and C3 did not bind mouse-PD-1-hFc1 (datanot shown). Anti-PD-1 antibody mAb7 bound mouse-PD-1-hFc1 weakly (datanot shown). All anti-PD-1 antibodies tested bound to hPD-1-hFc. Theseresults demonstrate that mAb7 is weakly cross-reactive with mouse PD-1whereas C1, C2, and C3 are not cross-reactive with mouse PD-1.

Example 8 Treatment of Cancer with Anti-PD-1 Antibodies

This is a prophetic example illustrating use of the anti-PD-1 antibodiesof the present invention for treating cancer.

Patients with histologically confirmed, previously untreated, measurablemetastatic colorectal cancer are selected for treatment with ananti-PD-1 antibody. Patients are assigned to one of two treatmentgroups: chemotherapy plus placebo or chemotherapy plus mAb7. A dynamicrandomization algorithm is utilized to achieve balance overall andwithin each of the following categories: study center, baseline ECOGperformance status (0 vs.≧1), site of primary disease (colon vs.rectum), and number of metastatic sites (1 vs.>1). The chemotherapytreatment is administered weekly for the first 6 weeks of each 8-weekcycle. Chemotherapy is continued until study completion (96 weeks) ordisease progression. mAb7 5 mg/kg or placebo is administered every 2weeks. Patients in the mAb7 arm who have a confirmed complete responseor experienced unacceptable toxicity as a result of chemotherapytreatment are allowed to discontinue chemotherapy and continue receivingmAb7 alone as first-line treatment. Only patients who are randomized tothe mAb7 group may receive mAb7 as a component of second-line treatment.After completing the study, patients are followed for any subsequenttreatment and survival every 4 months until death, loss to follow-up, ortermination of the study.

Patients will undergo an assessment of tumor status at baseline and atcompletion of every 8-week cycle using appropriate radiographictechniques, typically spiral CT scanning. Tumor response, orprogression, will be determined by both the investigator and anindependent radiology facility (IRF) utilizing the Response EvaluationCriteria in Solid Tumors. Therasse et al. (2000). The IRF assessmentwill be performed without knowledge of the treatment assignment orinvestigator assessment. In addition, patients will complete theFunctional Assessment of Cancer Therapy-Colorectal (FACT-C), Version 4,a validated instrument for assessing quality of life (QOL) in colorectalcancer patients, at baseline and prior to each treatment cycle untildisease progression. Ward et al. (1999) Qual. Life Res. 8: 181-195.

Safety is assessed from reports of adverse events, laboratory testresults, and vital sign measurements. Adverse events and abnormallaboratory results are categorized using the National Cancer InstituteCommon Toxicity Criteria (NCI-CTC), Version 2. Prespecified safetymeasures include four adverse events of special interest (hypertension,proteinuria, thrombosis, and bleeding).

The primary outcome measure is duration of overall survival. Secondaryoutcome measures include: progression-free survival, objective responserate (complete and partial), response duration, and change in the FACT-CQOL score. Survival duration is defined as the time from randomizationto death. For patients alive at the time of analysis, duration ofsurvival will be censored at the date of last contact. Progression-freesurvival is defined as the time from randomization to the earlier ofdisease progression or death on study, defined as death from any causewithin 30 days of the last dose of study drug or chemotherapy. Forpatients alive without disease progression at the time of analysis,progression-free survival will be censored at their last tumorassessment, or day 1 (the first day of study treatment) if nopostbaseline assessment was performed. In the analysis of objectiveresponse, patients without tumor assessments are categorized asnonresponders. Disease progression and response analyses are based onthe IRF assessments. Change in quality of life is analyzed as time todeterioration in QOL (TDQ), defined as the length of time fromrandomization to a the earliest of a ≧3-point decrease from baseline incolon-cancer specific FACT-C subscale score (CCS), disease progression,or death on study. TDQ will also be determined for the TOI-C (sum ofCCS, physical and functional well-being) and total FACT-C for changesfrom baseline.

Example 9 Treatment of Cancer with Anti-PD-1 Antibodies

This example illustrates use of the anti-PD-1 antibodies of the presentinvention for treating cancer.

The study in this example is a Phase 1, open-label, multi center,multiple-dose, dose escalation, safety, PK, and PD study of anti-PD-1monoclonal antibody mAb7 administered intravenously in previouslytreated adult patients with locally advanced or metastatic melanoma,squamous cell head and neck cancer (SCHNC), ovarian carcinoma, sarcoma,or relapsed or refractory classic Hodgkin's Lymphoma (cHL). The studyprotocol is summarized below in Table 16.

TABLE 16 Arms Assigned Interventions Arm 1: mAb7 0.5 mg/kg every 21 daysDrug mAb7 IV every 21 days Arm 1: mAb7 1.0 mg/kg every 21 days Drug mAb7IV every 21 days Arm 1: mAb7 3.0 mg/kg every 21 days Drug mAb7 IV every21 days Arm 1: mAb7 10 mg/kg every 21 days Drug mAb7 IV every 21 daysInclusion Criteria: —Histological or cytological diagnosis of locallyadvanced or metastatic melanoma, SCCHN, ovarian cancer, sarcoma, orrelapsed or refractory cHL: —Patient should have received at least 1 andno more than 5 prior lines of therapy for recurrent or metastaticdisease, including both standards of care and investigational therapies.—At least one measurable lesion as defined by RECIST version 1.1, or(for cHL) at least 1 fluordeoxyglucose positron emission tomography (FDGPET) avid (Deauville 4/5) measurable lesion >1.5 cm as defined byResponse Criteria for Malignant Lymphoma that has not previously beenirradiated. —For Part 1B expansion and all Part 2 cohorts: patient hasconsented to undergo a pre treatment and on treatment biopsy. —AdequateRenal, Liver, bone marrow function Exclusion Criteria—Active brain orleptomeningeal metastases. —Ocular melanoma—Active, known or suspectedautoimmune disease. Patients with vitiligo, type I diabetes mellitus,residual hypothyroidism due to autoimmune condition only requiringhormone replacement, psoriasis not requiring systemic treatment, orconditions not expected to recur in the absence of an external triggerare permitted to enroll. Diagnosis of prior immunodeficiency or organtransplant requiring immunosuppressive therapy, —For Part 2: priortreatment with a PD 1 or PD L1 antibody. —History of Grade ≧3 immunemediated AE (including AST/ALT elevations that where considered drugrelated and cytokine release syndrome) that was considered related toprior immune modulatory therapy (eg, immune checkpoint inhibitors, costimulatory agents, etc.) and required immunosuppressive therapy.

The number of patients with ORR (Objective Response Rate) will bemeasured at baseline and every six weeks until disease progression orunacceptable toxicity up to 24 months.

Example 9 Antagonistic Activity of Anti-PD-1 Antibodies in Human andCynomolgus Monkey Primary T Cells

This example illustrates the activity of anti-PD-1 antibodies in humanand cynomolgus monkey primary T cells,

In this study, the antagonistic activities of anti-PD-1 monoclonalantibody mAb7 were examined in vitro using a mixed lymphocyte reaction(MLR). Primary T cells were isolated from human and cynomolgusmonkey-peripheral blood mononuclear cells (PBMCs). Following mAb7exposure, cell proliferation and cytokine secretion were evaluated invitro under different activation conditions using a MLR for human andcynomolgus monkey and cytokine release assay using cynomolgusmonkey-blood activated with Staphylococcal enterotoxin B (SEB) superantigen.

Methods Human T Lymphocytes

Human buffy coat was purchased from Stanford Blood Center (Stanford,Calif.), diluted with phosphate buffered saline (PBS) and layered overFicoll for the isolation of PBMCs. The hu-PBMCs were washed 4 times withPBS and T lymphocytes were isolated using a human-specific Pan T-cellisolation kit with negative selection as described in the manufacturer'sprotocol (Miltenyi Biotec, San Diego, Calif.).

Cynomolgus Monkey T Lymphocytes

Fresh cynomolgus monkey-PBMCs were purchased from Bioreclamation IVT(New York, N.Y.) and washed twice with PBS. T lymphocytes were isolatedusing a non-human primate specific for pan T-cell isolation kit withnegative selection as described in the manufacturer's protocol (MiltenyiBiotec, San Diego, Calif.).

Generating Human Dendritic Cells Expressing High Levels of PD-L1

Human buffy coat was purchased from Stanford Blood Center (Stanford,Calif.), diluted with PBS and layered over Ficoll for the isolation ofhu-PBMCs. The hu-PBMCs were washed 4 times with PBS and cluster ofdifferentiation 14 (CD14⁺) monocytes were isolated using a humanspecific CD14 cell isolation kit with positive selection, as describedin the manufacturer's protocol (Miltenyi Biotec, San Diego, Calif.).Cells were then seeded at 5×10⁵ cells/mL in complete Roswell ParkMemorial Institute (RPMI) 1640 media supplemented with 10% fetal bovineserum (FBS) for 7 days. Cultures were supplemented with recombinanthuman (rh-) IL-4 (1000 U/mL) (R&D Systems, Minneapolis, Minn.) and withrh-granulocyte-macrophage colony-stimulating factor (GM-CSF) (rh-GMCSF)(500 U/mL) (R&D Systems, Minneapolis, Minn.) at Days 0, 2 and 5.Immature DCs were harvested, washed, and counted on Day 7. A sample ofeach preparation was tested for PD-L1 expression using r-phycoerythrin(RPE) labeled anti-hu-PD-L1 (eBioscience/Affymatrix, San Diego, Calif.)by flow cytometry using a LSRFortessa™ analyzer (BD Biosciences, SanJose, Calif.).

Generation of Cynomolgus Monkey Dendritic Cells Expressing High Levelsof PD-L1

Fresh cynomolgus monkey-PBMCs were purchased from Bioreclamation IVT(New York, N.Y.) and washed twice with PBS. CD14+ monocytes wereisolated using a non-human primate specific CD14 cell isolation kit withpositive selection as described in the manufacturer's protocol (MiltenyiBiotech, San Diego, Calif.). Cells were then seeded as 5×10⁵ cells/mL incomplete RPMI 1640 media supplemented with 10% FBS for 7 days. Cultureswere supplemented with rhIL-4 (1000 U/mL) (R&D Systems, Minneapolis) andrh-GMCSF (500 U/mL) (R&D Systems, Minneapolis, Minn.) at Days 0, 2, and5. Immature DCs were harvested, washed, and counted on Day 7. A sampleof each preparation was tested for PD-L1 expression using RPE-labeledanti-hu-PD-L1 (eBioscience/Affymatnx, San Diego, Calif.) by flowcytometry using a LSRFortessa™ analyzer (BD Biosciences, San Jose,Calif.).

Generation of JeKo-1-Luc-Green Fluorescent Protein Cell ClonesExpressing High Levels of Human PD-L1

The JeKo-1 cell line (a Mantle Cell Lymphoma) was purchased fromAmerican Type Culture Collection (ATCC, Manassas, Va.). TheJeKo-1-luc-2A-GFP cell line was produced at Pfizer (South San Francisco,Calif.) by a transduction process that used lentiviral particlesexpressing individually firefly luciferase (luc2A) and green fluorescentprotein (GFP) through a bicistronic system with a blasticidin marker(AMSBIO, LVP323, 1×10E⁷ particles per 200 μL) according to themanufacturer's protocol. JeKo-1 cells were pelleted and diluted to 1×10⁶cells/mL in RPMI with 20% FBS medium. Lentiviral particles were added tothe diluted cells at a ratio of 50 μL virus per 0.5 mL cells. Togenerate a JeKo-1 cell line expressing hu-PD-L1, hu-PD-L1 cDNA wascustom synthesized and cloned into generic expression vector (pcDNA3.1)by Life Technologies (San Diego, Calif.). A JeKo-1-Luc-GFP cell linestably expressing hu-PDL-1 was produced at Pfizer (South San Francisco,Calif.) by electroporation using the Amaxa® Nucleofector system (Lonza,Walkersville, Md.) and Kit V used according to the manufacturer'sprotocol (Lonza, Walkersville, Md.). Cells were then grown in thepresence of 250 μg/mL hygromycin for 2 weeks and then selected by cellsorting using BD FACSAria™ II cell sorter (BD Biosciences, San Jose,Calif.). The sorting of cells was conducted with the anti-hu-PD-L1antibody clone MIH-1 (Affymetrix/eBioscience, San Diego, Calif.) labeleddirectly with allophycocyanin (APC) label. Positive clones were expandedand tested for high PD-L1 expression using flow cytometry (LSRFortessa™analyzer, BD Biosciences, San Jose, Calif.). Clones that were generatedfrom single sorted cells and contained high levels of PD-L1 expressionwere selected.

Generation of Antibodies

mAb7 was generated in CHO cells (Pharmaceutical Sciences, Pfizer Inc,Saint Louis, Mo.) using Good Laboratory Practices (GLP) material (Lot NoSTL0005717) and provided in 20 mM histidine, 85 mg/mL sucrose, 0.2 mg/mLpolysorbate-80, 0.05 mg/mL disodium EDTA, pH 5.5 buffer. Endotoxin wasmeasured as ≦0.01 EU/mg.

The control antibody used in all in vitro assays was an anti-bovineherpes virus cloned into an IgG4-HG framework (the same framework asMAB7). The control antibody was generated at Pfizer (South SanFrancisco, Calif.), Lot No 4945, with ≦0.3 EU/mg endotoxin.

Two anti-hu-PD-1 antibodies were generated from sequences published inprevious patents and expressed in the IgG4-HG framework resembling theone used for MAB7. The antibodies were generated at Pfizer (South SanFrancisco, Calif.) and were designated as positive control 1 andpositive control 2 (Lot No 5053 and 4255, respectively). Endotoxinmeasured was ≦0.13 EU/mg and ≦0.056 EU/mg, respectively.

Human Assay Using Dendritic Cells Expressing High Levels of PD-L1

The protocol was adapted from Kruisbeek et al, 2004, with somemodifications. Differentiated primary hu-DCs were harvested on Day 7 andverified by flow cytometry for high levels of PD-L1 expression andco-stimulatory signals necessary for T-cell activation; markers includedCD80 and CD86 (antibodies purchased from BD Bioscience, San Jose,Calif.). Cells were counted and irradiated at 3000 radiation units(rads) using a RS2000 x-ray machine (Radsource, Brentwood, Tenn.) toprevent DCs from secreting cytokines but functioning only as Agpresentation support to the T cells. Thus, the outcome of the assay wasonly induced by T cells. At Day 7, freshly isolated hu-T cells fromallogenic donors were harvested. T cells were plated with irradiated DCsat a ratio of 10:1 (optimal assay conditions were determined as 2×10⁵ Tcells incubated with 2×10⁴ DCs in 200 μL cultures) in the presence ofdifferent concentrations of mAb7, negative and positive controlantibodies, or media alone (to evaluate the baseline reaction). Allconditions were plated in 96-well flat bottom tissue culture treatedplates (Fisher Scientific Pittsburgh, Pa.). Cells were cultured usingserum free X-vivo15 media (Lonza, Walkersville, Md.) to prevent humanserum variability between experiments. Cultures were incubated at 37° C.with 5% CO2 for 5 days. On Day 5, supernatants were collected andcytokine concentrations were measured using cytometric beads array (CBA)(BD Biosciences, San Jose, Calif.) according to the manufacturer'sprotocol. Data were acquired using flow cytometry (LSRFortessa™analyzer, BD Biosceinces, San Jose, Calif.) and data analysis wasperformed using BD FCAP Array Software Version 3.0 (BD Biosceinces, SanJose, Calif.). Proliferation was measured in parallel cultures by adding1 μCi of 3H methyl-titrated thymidine (Perkin Elmer, Waltham, Mass.) toeach well and further incubated for 16 to 18 hours. Cultures were thenharvested on deoxyribonucleic acid (DNA) incorporation filters (PerkinElmer, Waltham, Mass.), and tritiated thymidine incorporation providedan index of cell proliferation measured as counts per minutes (cpm)using the MicroBeta2 Machine (Perkin Elmer, Waltham, Mass.).

Human Assay Using JeKo1-PD-L1 Expressing Cell Line

This assay was performed using a 5:1 ratio of T cells to JeKo-1-PD-L1cell line was used since this ratio provided a more ideal approach tocapture cytokine secretion on Day 5. Thus, each well was incubated with2×10⁵ T cells with 4×10⁴ JeKo-1-PD-L1. On Day 5, supernatants werecollected and cytokine concentrations were measured using CBA (BDBiosciences, San Jose, Calif.) according to the manufacturer's protocol.This cell line expresses a very modest amount of co-stimulatorymolecules (CD80 and CD86), and thus, enhanced proliferation is very mildfollowing antibody treatment. In contrast, cytokine secretion, includingIL-2, is robust, thereby allowing the measurement of cytokine secretionfollowing mAb7 treatment and not T-cell proliferation.

Cynomolgus Monkey Assay Using Dendritic Cells Expressing High Levels ofPD-L

Differentiated DCs were harvested on Day 7 and verified by flowcytometry (using LSRFortessa™ analyzer, BD Biosceinces, San Jose,Calif.) for high PD-L1 expression and co-stimulatory signals necessaryfor T-cell activation; markers included CD80 and CD86 (antibodies fromBD Bioscience, San Jose, Calif.). Cells were counted and irradiated at3000 rads using RS2000 X-ray machine (Radsource, Brentwood, Tenn.).Freshly-isolated cynomolgus monkey T cells from allogenic donors wereharvested. T cells were plated with irradiated DCs using a ratio of 10:1T-cells to DCs (optimal assay when 2×10⁵ T cells were incubated with2×10⁴ DCs in 200 μL culture) in the presence of different concentrationsof mAb7, negative and positive control antibodies, or media alone toevaluate the baseline reaction. All conditions were plated in 96-wellflat-bottom-tissue culture-treated plates (Fisher Scientific,Pittsburgh, Pa.). Cells were cultured using serum free X-vivo15 media(Lonza, Walkersville, Md.) to prevent serum variability betweenexperiments. Cultures were incubated at 37° C. with 5% CO2 for 5 days.On Day 5, supernatants were collected and cytokine concentrations weremeasured using CBA (BD Biosciences, San Jose, Calif.) according to themanufacturer's protocol. Data were acquired using flow cytometry(LSRFortessa™ analyzer, BD Biosceinces San Jose, Calif.), and dataanalysis was performed using BD FCAP Array Software Version 3.0 (BDBiosciences, San Jose, Calif.). At the same time and in similarcultures, 1 μCi of 3H methyl-titrated thymidine (Perkin Elmer, Waltham,Mass.) was added to each well; cells were further cultured for 16 to 18hours to measure proliferation. Cultures were harvested on DNAincorporation filters Glass Printed filtermate A (Perkin Elmer, Waltham,Mass.) and tritiated thymidine incorporation, providing an index of cellproliferation, was measured as cpm using MicroBeta2 Machine (PerkinElmer, Waltham, Mass.).

Cytokine Release from Cynomolgus Monkey Blood Induced by SuperantigenStimulation (Staphylococcal Enterotoxin B)

Whole blood from cynomolgus monkey was collected; 225 μL of blood werealiquoted into tissue-culture treated 96 well plates (Fisher Scientific,Pittsburgh, Pa.). Samples were incubated in duplicate at 37° C. in 5%CO2 in the presence of mAb7 or an isotype-matched negative controlantibody at concentrations ranging from 0.1 to 100 μg/mL. One hour afterthe antibody addition, samples were stimulated with 0.1 μg/mL SEB (ToxicTechnologies, Sarasota, Fla.) and cultures were incubated for 3 days. OnDay 3, plasma was harvested, pooled, and frozen at −80° C.Concentrations of IFN-γ, IL-2, and TNF-α in thawed serum samples weremeasured in duplicate according to the manufacturer's protocol using MSDimmunoassay plates (Meso Scale Diagnostics, Rockville, Md.) and an MSDReader (Model 1200) with MSD Discovery Workbench software (Version4.0.12). The means of the duplicates were reported.

Results

Antagonistic Activity of mAb7 on Human Primary T Cells

When primary human T cells were activated in MLR with allogenic hu-DCsexpressing PD L1, mAb7 increased T-cell proliferation (measured bytritiated thymidine incorporation) and T-cell activation (measured bypro-inflammatory cytokine secretion) in a dose-dependent manner.Treatment of T-cells with mAb7 (10 μg/mL) resulted in an increase inT-cell proliferation of up to 2.5-fold over treatment with a negativecontrol antibody (10 μg/mL). IFN-γ and TNF-α levels were increased up to8- and 5-fold, respectively, when compared to negative control antibody.IFN-γ increase was superior to the TNF-α increase. IL-2 expression wasnot detected in these cultures. When primary human T cells wereactivated in MLR, using the tumor cell line JeKo 1 PD-L1 as allogenicantigen presenting cells, mAb7 induced a dose-dependent increase ofIFN-γ(up to 2.5-fold), TNF-α (up to 2-fold), and IL-2 (up to 5-fold)secretion as compared to negative control antibody. The effect of mAb7in this assay resembled the data obtained with both positive controlantibodies. Increased T-cell proliferation was not observed under theseconditions. Cell proliferation was minimal with a weak signal providedby CD80 and CD86 in comparison to MLR mediated by primary DCs. IL-10,IL-4, IL-17A and IL-6 were minimal to non-detected in all assaysdescribed above.

Antagonistic Activity of mAb7 on Cynomolugs Monkey Primary T Cells

The binding affinity of mAb7, when in solution, to hu-PD-1 andcynomolgus monkey PD-1 were very similar in the kinetic exclusion assay(KinExA) (KD=23 and 28 pM for human and cynomolgus monkey PD-1,respectively). The EC50 of mAb7 on cells expressing human PD-1 andcynomolgus monkey PD-1 was also similar. In a MLR functional assay thatused T cells and DCs isolated from different cynomolgus monkeys, mAb7induced T cell proliferation and activation in a dose dependent manner(measured by tritiated thymidine incorporation). This effect was alsoobserved with positive control (antibody 1 and antibody 2) but not withthe negative control antibody. mAb7 also enhanced cytokine secretion(ie, IFN-γ and TNF-α, up to 5-fold and 3-fold, compared to treatmentwith negative control antibody. IL-2 expression was not detected inthese cultures. In a different cytokine-release assay using cynomolgusmonkey whole blood stimulated with 0.1 μg/mL of SEB superantigen for 3days, mAb7 (0.1-100 μg/mL) induced greater IFN-γ, IL-2, and TNF-αsecretion when compared with the negative control antibody.

MLR studies were used to create an in vitro setting resembling tumormicroenvironment where T cells are activated and expressing high levelsof PD-1 in the presence of allogeneic DCs or tumor cells expressingPD-L1. PD-1/PD-L1 interaction inhibits further T-cell proliferation andcytokine release Addition of anti-PD-1 antibodies in these MLRs restoredT-cell activation and increased proliferation and cytokine secretion,especially IFN-γ, due to block of the PD-1/PD-L1 axis.

mAb7 accelerated the proliferation and secretion of IFN-γ, TNF-α, andIL-2 by human T cells when cultured with allogenic cells expressing highlevels of PD-L1 (DCs or JeKo 1 PD-L1 cell line). In contrast, thenegative control antibody, which proved similar to media alone, did notenhance these effects. Similarly, mAb7 enhanced T cell proliferation,IFN-γ and TNF-α secretion in the cynomolgus monkey-MLR system, as wellas enhanced cytokine release from cynomolgus monkey whole blood usingsuper antigen SEB.

These results demonstrate that anti-PD-1 antibody mAb7 enhanced T-cellproliferation and pro-inflammatory cytokine secretion includinginterferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), andinterleukin (IL)-2. These activities were observed both in primary humanand cynomolgus monkey T cells.

Example 10 Effect of Anti-PD-1 Antibodies in Graft Versus Host Disease

This example illustrates the effect of anti-PD-1 antibodies on T-cellactivation and expansion in vivo using a xeno-aGvHD model in NSG micetransferred with hu-PBMCs.

In this study, the effect of anti-PD-1 antibody mAb7 on T-cellactivation and expansion in vivo was studied in a xeno acute graftversus host disease (aGvHD) model in non-obese diabetic (NOD),severe-combined immunodeficiency (SCID), interleukin 2 receptor gammanull (IL2rγ^(nuil)) (NSG) mice using hu-peripheral blood mononuclearcells (PBMCs).

Immunocompromised NSG female mice (5 per group) aged 8 to 10 weeks(formal name, NOD, Cg-Prkdc^(scid)II2rg^(tm1Wjl)/SzJ) were purchasedfrom the Jackson Laboratory (Bar Harbor, Me.). All animals were housedin a pathogen free facility at Pfizer (South San Francisco, Calif.) inaccordance with the Institutional Animal Care and Use Committee (IACUC).

Human buffy coat was purchased from Stanford Blood Center (Standord,Calif.), diluted with phosphate buffered saline (PBS) and layered overFicoll for the isolation of PBMCs. The PBMCs were washed 2 times withPBS. Red blood cells (RBCs) were lysed using ammonium-chloride-potassium(ACK) lysing buffer as indicated in the manufacturer's protocol (LifeTechnologies; San Diego, Calif.). After the RBC lysis, cells were washedonce more and diluted in PBS at 5×10⁷ cells per mL.

For induction of xeno-aGvHD, NSG mice were injected intravenously viatail vein with hu-PBMCs. Each mouse received 1×10⁷ cells in 200 μL ofPBS. In all experiments, mice were weighed 3 times weekly and monitoredfor the appearance of xeno-aGvHD-like symptoms including weight loss,hunched posture, ruffled fur, reduced mobility, and in some cases,diarrhea. Mice were euthanized after loss of 20% body weight or the lossof 1 g/day over 2 days; this time point was recorded as the survivaltime. mAb7, negative control antibody, and positive control antibodieswere generated as described above in Example 9.

NSG mice were treated with negative control antibodies, positive controlantibodies, or mAb7, at doses range between 0.1-10 milligrams perkilograms (mg/kg). Antibodies were administered to mice using theappropriate vehicle on Day 2 and Day 8 post hu-PBMC transfer. The routeof antibody administration was intraperitoneal (ip).

Peripheral blood, spleens, and livers were harvested from micetransplanted with hu-PBMCs. Single-cell suspensions from each organ wereprepared as follows: Peripheral blood was collected and RBCs were lysedusing ACK-lysing buffer and washed with PBS. Spleens and livers weremechanically homogenized using the back of a syringe plunger to maceratethe cells through a 70 μM filter and washed once with PBS. RBCs werelysed and cells were washed 2 more times and macerated again through the70 μM filter. At this stage, spleen cells were ready. For isolatingleukocytes from liver, single cell suspensions were layered usingPercoll gradient at 30% to 80% (Percoll® Plus, GE HealthcareBio-Sciences, Pittsburgh, Pa.) and subjected to high-speedcentrifugation. Leucocytes were harvested from the intermediate layerand washed twice with PBS. All single cell suspensions were counted and1×10⁶ cells from each sample were used for fluorescence-activated cellsorting (FACS). For human cytokine secretion assays, mouse CD45⁺ cellswere depleted using a mouse specific CD45 cell isolation kit by positiveselection, as described in the manufacturer's protocol (Miltenyi Biotec,San Diego, Calif.), to ensure that cytokine-secretion was from humanimmune cells alone. Cells were counted, and 1×10⁶ cells from each samplewere used in the assay.

Single cell suspensions from peripheral blood, spleens, and livers wereobtained from xeno-aGvHD mice. A total of 1×10⁶ cells per sample wereincubated for 30 minutes at 4° C. under protection from light with amixture of appropriate fluorescently-labeled monoclonal antibodies inFACS buffer including 1×PBS containing 2% fetal bovine serum (FBS), thencells were washed with FACS buffer. For intracellular staining of Kiel67 protein (Ki67, to measure proliferating cells), the cells were fixedafter surface staining using Intracellular Fixation & PermeabilizationBuffer Set (Affymetrix/eBioscience, San Diego, Calif.) according to themanufacturer's protocol. Intracellular staining was performed for 30minutes at 4° C. in the dark. At the end of staining, samples werewashed and subjected to multicolor flow cytometry using BD LSR Fortessa™cell analyzer (BD Biosciences, San Jose, Calif.). Data analyses weredone using FlowJo software (FLOWJO LLC, Ashland, Oreg.). Antibodies usedin different combinations for recognition of the cell surface moleculeswere: anti-hu-CD45 Pacific Blue (PB) or Amcyan, anti-mouseCD45-Brilliant Violet (BV)-711, anti-hu-CD3-PerCPCy5.5, anti-hu-CD8phycoerythrin-Cy7 (PE-Cy7) or BV-786, anti-hu-CD4-fluoresceinisothiocyanate (FITC) or BV-650, anti-hu-PD-1 (done EH12.1) BV-786 orPE, anti-hu-PD-1 (clone MIH-4) PE or FITC, anti-hu-Ki67 allophycocyanin(APC) (all antibodies from BD Biosciences, San Jose, Calif.),anti-hu-PD-L1 (PE-Affymatrix-eBioscience, San Diego, Calif.) and bluefluorescent reactive dye for Live/Dead staining (Life Technologies,Grand Island, N.Y.).

Single cell suspensions of human CD45 enriched population from spleensand livers were obtained from xeno-aGvHD mice (after mouse CD45depletion). A total of 1×10⁶ cells per sample was incubated in flatbottom tissue culture treated 96-well plates (Fisher Scientific;Pittsburgh, Pa.) in 200 μL X-vivo 15 media. Cells were then leftunstimulated or stimulated with phorbol myristate acetate (PMA) 10 ng/mLand ionomycin (iono) 125 ng/mL, as the low stimulation condition or PMA50 ng/mL and ionomycin 1 μg/mL as the high stimulation condition (bothobtained from Sigma-Aldrich, Saint Louis, Mo.). Cultures were incubatedfor 8 hours at 37° C. in 5% CO₂ to ensure maximal cytokine secretion.Supernatants were collected and human cytokine concentrations weremeasured using cytometric beads array (CBA) specific for human cytokines(BD Biosciences, San Jose, Calif.) according to the manufacturer'sprotocol. Data were acquired using flow cytometry (LSRFortessa™analyzer, BD Biosciences, San Jose, Calif.), and data analysis wasperformed using FCAP Array™ Software Version 3.0 (BD Biosciences, SanJose, Calif.). Hu-IFN-γ, hu-TNF-α, and hu-IL-2 bead array kits werepurchased from BD Biosciences (San Jose, Calif.), and it was confirmedthat they did not cross-react with mouse cytokines.

All analyses involved a comparison of means using the independent-samplet-test or 2-way ANOVA using Graphpad Prism (Graphpad Software, SanDiego, Calif.). Values of p≦0.05 were considered statisticallysignificant. Engraftment data are depicted in figures as meanconcentrations including the standard error of the mean (sem).

Treatment of NSG immunocompromised mice with anti-PD-1 antibody mAb7accelerated body weight loss (Table 17) and induced other disease signsthat are commonly seen in this model, such as hunched posture, ruffledfur, decreased mobility, and in some cases, diarrhea. In this model,body weight loss is expected to accelerate between Day 20 and Day 30post transfer (see, e.g., Schroeder and DiPersio, 2011). Becausetreatment with mAb7 and positive controls 1 and 2 accelerated xeno-aGvHDsymptoms, mice had to be euthanized at earlier time points and asurvival curve could not be obtained; therefore, body weight loss wasthe primary outcome measurement. In the first experiment, mice weretreated with 0.1, 1, and 10 mg/kg of mAb7 or negative control antibodyat 10 mg/kg. In Table 17, body weight loss was calculated by normalizingthe body weight differences between the treated groups at Day 23relative to Day 0. Values indicate an average of 5 mice per group±sem.

TABLE 17 Body weight Body weight Body weight Body weight TreatmentGroups Day 0 Day 14 Day 23 # per group loss Negative control 20.68 ±0.36 22.00 ± 0.36 20.88 ± 1.11 5   0% mAb: 10 mg/kg mAb7: 10 m/kg 20.78± 0.36 17.76 ± 0.57 17.74 ± 0.86 5   15% mAb7: 1 mg/kg 21.52 ± 0.3619.26 ± 0.35  17.7 ± 0.58 5   15% mAb7: 0.1 mg/kg  20.5 ± 0.78 19.78 ±1.38 19.22 ± 1.95 5 7.95%

Mice were treated with the indicated antibodies on Day 2 and Day 8post-hu-PBMC transfer. While body weight loss became apparent in thecontrol group on Day 23, body weight loss and disease progression weredetected in individual mice from all anti-PD-1 antibody mAb7 treatedgroups starting Day 10-11 post hu-PBMCs transfer. In both the 1 mg/kgand 10 mg/kg treated groups body weight loss reached significancebetween Days 14 to 23, when compared to the negative control treatedgroup (p≦0.0001). On Day 23, a 15% weight loss was detected in the 1mg/kg and 10 mg/kg treated groups and a 7.9% weight loss was detected inthe 0.1 mg/kg treated group (Table 17).

FACS analyses of peripheral blood, liver, and spleen demonstrated anincrease in the percentage of hu-CD45 positive cells and hu-CD3 positiveT cells in the mAb7 treated group versus the control group.Representative data from spleens also showed increase in hu-CD8 positiveT-cell counts, and hu-CD4 positive T-cell counts (but to a lesserextent) in mAb7 versus the negative control antibody treatment group.Similar increase was also noted in hu-T cell counts in the liver andblood. To assess whether the increase in T cells was due to blockade ofPD-1 through binding of mAb7 to human T cells, the T cells were stainedwith a commercial antibody for PD-1 (clone EH12.1) that competes withbinding of mAb7. mAb7-treated lymphocytes from all organs (analyzed byFACS) showed no binding to EH12.1. To confirm that T cells treated withmAb7 still express PD-1, T cells were stained with a differentanti-hu-PD-1 clone (done MIH4) that partially competes with mAb7 forbinding to T cells. Results showed that T cells treated with mAb7partially bound MIH4; thus, indicating that PD-1 was expressed ontreated T cells and that PD-1 blockade by mAb7 was evident (data notshown, maintained in Pfizer internal records). No detectable changeswere observed for hu-PD-L1 expression in the spleen, on T cells (hu-CD3positive), or on non-hu-T cells (hu-CD3 negative). Ki67, a marker forlymphocyte proliferation, was elevated in hu-CD3 positive cells from thePF-0681591-treated group versus the negative control treated group inthe spleen middle panel). Similar results were seen in the blood andliver.

To examine the effects of mAb7 on cytokine release during xeno-aGvHD,hu-CD45 positive cells (from livers and spleens) were further isolatedfrom mouse CD45 positive cells. These lymphocytes were then treated witha mixture of PMA and ionomycin (in 2 concentrations: low versus high) orleft untreated for 8 hours at 37° C. Cytokine secretion was measured inthe supernatants by CBA (Table 18). No detectable cytokines wereobtained without stimulation (Table 18). Following mAb7 treatment,hu-IFN-γ, hu-IL-2, and hu-TNF-α were elevated in human lymphocytesisolated from both mouse spleen and liver compartments compared to thecontrol groups. Under weak ex vivo stimulation conditions, mAb7 treatedT cells increased hu-IFN-γ secretion to levels that were significantlyhigher than those of T cells isolated from the negative control group(p<0.05). Under strong ex vivo stimulation conditions, all cytokineswere induced in all groups but further increased in mAb7 treated T cellscompared to T cells isolated from negative controls. Table 18 shows datacollected from 5 different mice in each group. In Table 18, *p<0.05,**p<0.01 in unpaired t-test comparing mAb7 versus negative controlgroup; aGvHD=Acute graft versus host disease; Hu=Human; IFN-γ=Interferongamma; IL-2=Interleukin-2; Ino=Ionomycin; N=Number, ns=Not significant:P=P value; PMA=Phorbol myristate acetate; TNFα=Tumor necrosis factoralpha; Xeno=Xenogeneic.

TABLE 18 Ex Vivo Stimulation Treatment In Vivo Organ-Hu Conditions Hu-negative control Statistics Lymphocyte (PMA/Iono) Cytokine mAb7 mAbUnpaired Isolated (ng/mL) pg/mL 10 mg/kg 10 mg/kg N t-test Liver WeakIFN-γ  414.1 ± 59.4  163.9 ± 43.41  5 **P < 0.05 Spleen (10/125  2599.96± 863.45  503.9 ± 111.9  5  *P < 0.01 Liver ng/mL) IL-2   783.1 ± 776.2619.41 ± 5.29 5 ns Spleen  1813.98 ± 840.26 14.26 ± 1.40 5 ns Liver TNF-α 80.75 ± 60.45  9.92 ± 2.55 5 ns Spleen  368.81 ± 209.73  14.1 ± 2.51 5ns Liver Strong IFN-γ  3838.65 ± 178.84 2931.26 ± 556.98 5 ns Spleen(50/1000  3084.1 ± 204.1  2523.9 ± 141.2 5 ns Liver ng/mL) IL-2  7759.44± 809.24 4565.26 ± 1240.8 5 ns Spleen 16735.57 ± 1417   13204.5 ± 1834.35 ns Liver TNF-α  1611.9 ± 150.82  758.1 ± 188.2  5 **P < 0.01 Spleen 2842.6 ± 224.2   2311.7 ± 281.36 5 ns Liver None IFN-γ   3.1 ± 1.25 4.6 ± 0.96 5 ns Spleen (0/0  3.94 ± 0.97  4.86 ± 1.16 5 ns Liver ng/mL)IL-2   3.9 ± 0.14  4.66 ± 0.41 5 ns Spleen  3.23 ± 0.77  4.39 ± 0.55 5ns Liver TNF-α   0.1 ± 0.09  0.3 ± 0.2  5 ns Spleen   0.4 ± 0.15 0.27 ±0.3 5 ns

These results demonstrate that treatment of xeno-aGvHD with anti-PD-1antibody mAb7 accelerated xeno-aGvHD development in NSG mice, asmeasured by body weight loss, T-cell proliferation, and increasedcytokine secretion including interferon-gamma (IFN-γ) and interleukin-2(IL-2).

Example 11 Characterization of Anti-PD-1 Antibodies

This example illustrates binding affinity, specificity, and ligandblocking activity of anti-PD-1 antibody mAb7 on cells expressing surfacereceptor PD-1.

mAb7, negative control antibody, and positive control antibodies weregenerated as described above in Example 9. Anti-hu-PD-1 antibody cloneEH12.1, primary labeled with phycoerythrin (PE) or Brilliant Violet(BV)-786 and isotype control antibodies labeled with the same dyes werepurchased from BD Biosciences (San Jose, Calif.). PE-labeled anti-mousePD-1 clone J43 and anti-hamster IgG isotype control antibody werepurchased from Affymetrix/eBiosciense (San Diego, Calif.). Biotinylatedhu-PD-L1 and biotinylated hu-PD-L2 (CD273), were obtained from ACROBiosystems (Newark, Del.). Cynomolgus monkey PD-L1 and PD-L2 werepurchased from Creative BioMart® Recombinant Proteins (Shirley, N.Y.),and both were labeled in-house with Alexa Fluor® 647 dye using an AlexaFluor® 647 protein labeling kit (LifeTechnologies, San Diego, Calif.) asinstructed by the manufacturer's protocol. Cynomolgus monkey PD-L1 andPD-L2 were tested for binding to the cynomolgus monkey PD-1 expressingcell line. Rat- and mouse-PD-L1-Fc-Tags (Fc region of human IgG1 at theC-terminus) were purchased from Creative BioMart® Recombinant Proteins(Shirley, N.Y.). Detection of biotinylated PD-L1 and PD-L2 was achievedusing streptavidin PE or allophycocyanin (APC) (Affymetrix/eBiosciense,San Diego, Calif.). Detection of rat or mouse-PD-L1 was achieved usingAPC labeled Fc gamma (Fcγ) Fragment Specific donkey-anti-humanaffiniPure F(ab′)₂ IgG (Jackson ImmunoResearch Laboratories Inc, WestGrove, Pa.).

Vectors for Transient Transfection

Hu-PD-1 expression plasmid (in pCMV6-Entry vector) was purchased fromOriGene Technologies, Inc (Rockville, Md.), Catalog NoRC210364/Accession No NM_005018.

Mouse-PD-1 expression plasmid (in pCMV6-Entry vector) was purchased fromOriGene Technologies, Inc (Rockville, Md.), Catalog NoMR227347/Accession No NM_008798. Cynomolgus monkey-PD-1 was codonoptimized and synthesized by Life Technologies (San Diego, Calif.), LotNo 1482149/Accession No EF443145. It was cloned into a proprietaryPfizer (San Francisco, Calif.) cytomegalovirus (CMV)-based expressionplasmid, in frame with a mouse kappa secretory signal sequence whichadded a C-terminal FLAG tag to the C-terminus.

Rat-PD-1 deoxyribonucleic acid (DNA) was custom synthesized according tothe sequence from Accession No NM_001106927 and cloned into BamHI-NotIsites of expression vector pEF1V5-His from LifeTechnologies (San Diego,Calif.), Lot No1598305.

PD-1 Expression Vectors for Stable Transfection

Hu-PD-1 DNA was custom synthesized according to the sequence fromAccession No NM_005018 and cloned into BamHI-NotI sites of InVitroGenexpression vector pEF1V5-His B; Lot No 513478.

Cynomolgus monkey-PD-1 DNA was custom synthesized according to theAccession No EF443145 and cloned into BamHI-NotI sites of InVitroGenexpression vector pEF1V5-His B, Lot No 1482149.

Mouse-PD-1 DNA was custom synthesized according to the Accession NoNM_008798 and cloned into BamHI-NotI sites of InVitroGen expressionvector pEF1V5-His B, Lot No 1513476.

All vectors were synthesized and cloned into InVitroGen expressionvector pEF1V5-His B by Life Technologies (San Diego, Calif.). Allvectors contain the complete PD-1 sequence including extracellulardomain, membrane domain, and cytosolic domain. All vectors encoded aV5-6His epitope tag at the C-terminus of PD-1. The neomycin resistancegene was included in each vector for selection using G418 sulfateantibiotics.

Transient Transfection Using HEK-293T Cell Line

HEK-293T cells were purchased from American Type Culture Collection(ATCC®, Manassas, Va.), and cells were maintained in Dulbecco's ModifiedEagle's medium (DMEM) (Coming CellGro, Manassas, Va.) supplemented with10% fetal bovine serum (FBS) and 1× Penicillin/Streptomycin (Pen/Strep)and grown in 6% carbon dioxide (CO₂) at 37° C. One day prior totransfection, cells were trypsinized and plated at 4×10⁶ cells per T75flask (Fisher Scientific, Pittsburgh, Pa.). On the day of transfection,pre-warmed, antibiotic-free growth media replaced the old media andcells were further incubated for 2 hours at 37° C. in 6% CO2. Expressionvector or empty vector (10 μg) was added to 1.5 mL OptiMEM media (LifeTechnologies, San Diego, Calif.). Lipofectamine 2000 Reagent (20 μl)(Life Technologies, San Diego, Calif.) was then added to another 1.5 mlOptiMEM. The plasmid OptiMEM and Lipofectamine OptiMEM tubes were thenmixed together and incubated at room temperature for 25 minutes. TheOptiMEM mixture was then added drop-wise to the appropriate cultureflask, and the flask was left overnight at 37° C. in 6% CO2. Thefollowing day, the media was removed from the flask and replaced withcomplete growth media. Forty-eight (48) hours following transfection,cells were harvested using the StemPro-Accutase (Life Technologies, SanDiego, Calif.), thereby, allowing cells to be gently removed from theculture surface without affecting PD-1 surface expression. Cells werethen subjected to antibody binding and fluorescence-activated cellsorting (FACS) analyses.

Stable Transfection Using Jurkat Cell Line

The Jurkat cell line, (clone E6-1-TIB-152™, ATCC®, Manassas, Va.), wasused to stably express hu- and cynomolgus monkey-PD-1. Cell lines weregenerated using electroporation via Amaxa necluotransfector system(Lonza, Walkersville, Md.). Transfections were performed at Pfizer (SanFrancisco, Calif.) using a Kit V as instructed by the manufacturer'sprotocol (Lonza, Walkersville, Md.). Cells were maintained in growthmedia Roswell Park Memorial Institute (RPMI)-1640 supplemented with 10%FBS and 1×L-glutamine (LifeTechnologies, San Diego, Calif.) at 37° C. in5% CO2 at a density between 0.3 to 1.0×10⁶ cells/ml. For each of thevectors, 2 μg/mL was used to transfect 2×10⁶ cells. After thetransfection, cells were grown in the presence of G418 Sulfate (600μg/mL) for 2 weeks for selection and maintenance of thestably-transfected cells. To prevent long term contamination, 1×Pen/Strep was added to the media. Single cell clones were selected byculturing the cells in a dilution of 1:200 (1 cell in 200 μL completemedia supplemented with G418 sulfate antibiotics). To select for clonesexpressing high hu- or cynomolgus monkey-PD-1, PE-labeled-anti-hu-PD-1clone EH12.1 was used in flow cytometry assays. Samples were collectedusing BD LSRFortessa™ cell analyzer (BD Biosciences, San Jose, Calif.).Data analyses and mean fluorescence intensity (MFI) were calculatedusing Flowjo software (FLOWJO LLC, Ashland, Oreg.). Cell lines with highMFI were chosen for assay development.

HEK-293T cells transfected with hu-, cynomologus monkey-, mouse- orrat-PD-1 vectors, or empty vector were harvested at 48 hours posttransfection. Prior to binding with mAb7, cells from each specifictransfection were tested with commercial antibodies or ligands to ensurethat the appropriate PD-1 receptor was highly expressed on the cellsurface. For human and cynomolgus monkey, cross reactive commercialanti-PD-1 antibody (clone EH12.1) was used. For mouse, a commercialanti-mouse-PD-1 antibody (clone J43) was used. For rat, a rat-PD-L1ligand was used (since there was no commercial rat cross-reactiveanti-PD-1 antibody available). After appropriate PD-1 expression wasconfirmed, cells were counted, and 2×10⁵ cells were incubated with hu-FcReceptor Binding Inhibitor Functional Grade mixture (Fc-γ receptor blockat 1 μg/1×10⁸ cells, Affymetrix/eBiosciense, San Diego, Calif.) for 20minutes and blue fluorescent reactive dye, used for Live/Dead staining,was added to the mixture (Life Technologies, San Diego, Calif.). Serialdilutions of mAb7 or negative control (1-0.00001 μg/mL) antibody wereadded to the cell mixtures and then allowed to incubate on ice for 1hour. Cells were then washed andAPC-labeled-donkey-anti-human-affiniPure F(ab′)₂ Fragment IgG, FcγSpecific (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, WestGrove, Pa.) was added to the mixture, and cells were, thereafter,incubated on ice for another 30 minutes. Data were acquired on the BDLSRFortessa™ cell analyzer (BD Biosciences, San Jose, Calif.), andanalyzed using Flowjo software (FLOWJO LLC, Ashland, Oreg.).

Stable Jurkat cell clones that were transfected with either the hu-,cynomolgus monkey-, or mouse-PD-1 receptor were generated. PD-1 Cellclones chosen for these assays from each species expressed similaramounts of PD-1 receptors (˜400,000 receptors/cell; receptors werequantified previously during the generation of these cell lines). Afterappropriate PD-1 expression was confirmed, cells were counted, and 2×10⁵cells were incubated with hu-Fc Receptor Binding Inhibitor FunctionalGrade (Fc-γ receptor Block at 1 μg/1×10⁶ cells, Affymetrix/eBiosciense,San Diego, Calif.) for 20 minutes and blue fluorescent reactive dye,used for Live/Dead staining, was added to the mixture (LifeTechnologies, San Diego, Calif.). Serial dilutions (1:3) of mAb7,positive control 1, positive control 2, or negative control antibodies(using an IgG4 framework) were added to the cell mixture and thenallowed to incubate on ice for 1 hour. Cells were then washed andAPC-labeled-donkey-anti-human-affiniPure F(ab′)₂ Fragment IgG, FcγSpecific (1:100 dilution) (Jackson ImmunoResearch Laboratories Inc, WestGrove, Pa.) was added to the mixture and cells were then allowed toincubate on ice for another 30 minutes. Data were acquired on the BDLSRFortessa™ cell analyzer (BD Biosciences, San Jose, Calif.). Analyseswere done using Flowjo software (FLOWJO LLC, Ashland, Oreg.) andgeometrical means were calculated. EC₅₀ values were calculated usingGraph Pad Prism (Log agonistic versus response [binding]).

Activated T cells expressing high levels of PD-1 receptors weregenerated. Appropriate PD-1 expression was confirmed for the T cells,obtained from each species, using commercial reagents (anti-hu-PD-1clone EH12.1 for human and cynomolgus monkey, anti-mouse-PD-1 done J43for mouse-PD-1, and rat-PD-L1 for rat-PD-1). Cells were counted and1×10⁶ cells were incubated with hu-Fc Receptor Binding InhibitorFunctional Grade (Fc-γ receptor Block at 1 μg/1×10⁶ cells,Affymetrix/eBiosciense, San Diego, Calif.) for 20 minutes and bluefluorescent reactive dye, used for Live/Dead staining, was added to themixture (Life Technologies, San Diego, Calif.). Serial dilutions of mAb7(dilution 1:3) were added to the cell mixture and then allowed toincubate on ice for 1 hour. Cells were then washed beforeAPC-labeled-donkey-anti-human-affiniPure F(ab′)₂ Fragment IgG, Fcγspecific secondary antibody (1:100 dilution) (Jackson ImmunoResearchLaboratories Inc, West Grove, Pa.) was added, and cells were allowed toincubate on ice for 30 minutes. Data were acquired on the BDLSRFortessa™ cell analyzer (BD Biosciences, San Jose, Calif.). Dataanalyses were done using Flowjo software (FLOWJO LLC, Ashland, Oreg.).Geometrical means were counted and EC₅₀ values were calculated using PadGraph Prism (Log agonistic versus response [binding]).

Stable Jurkat cell clones expressing high levels of hu-PD-1 were chosenfor the assay. Twenty (20) μg/mL of either biotinylated-hu-PD-L1 orbiotinylated-hu-PD-L2 saturated all PD-1 receptors on this cell line(after previse binding titration assays). Therefore, this concentrationwas chosen as the optimal concentration in our studies. Hu-PD-1expressing Jurkat cells (2×10⁵) were incubated with hu-Fc ReceptorBinding Inhibitor Functional Grade (Fc-γ receptor block at 1 μg/1×10⁶cells, Affymetrix/eBiosciense, San Diego, Calif.) for 20 minutes, andblue fluorescent reactive dye, used for Live/Dead staining, was added tothe mixture as well (Life Technologies, San Diego, Calif.).Biotinylated-hu-PD-L1 or biotinylated-hu-PD-L2 were added to the cellsat 20 μg/mL, immediately followed by addition of serial dilutions ofmAb7, positive control 1, positive control 2, or negative controlantibodies (all in IgG4 backbone and in 1:3 serial dilutions). Cellswere allowed to incubate on ice for 1 hour. Cells were then washed andPE Streptavidin (1:100 dilution) (Affymetrix/eBiosciense, San Diego,Calif.) was added and the cells allowed to incubate on ice for 30minutes. Data were then acquired on the BD LSRFortessa™ cell analyzer(BD Biosciences, San Jose, Calif.) and analyzed using Flowjo software(FLOWJO LLC, Ashland, Oreg.) and geometrical means were calculated. TheIC50 values were calculated using Graph Pad Prism (Log inhibitor versusresponse [binding]).

Jurkat cells were stably transfected with high levels of hu- orcynomolgus monkey-PD-1 receptors. Cells expressing cynomolgusmonkey-PD-1 receptors (˜400,000 receptors/cell) were chosen for thisassay. Cynomolgus monkey-PD-L1 and PD-L2 binding was tested using thesecells and results showed that 20 μg/mL of either cynomolgus monkey-PD-L1or PD-L2 was enough to saturate all PD-1 receptors based on thegeometrical mean tested for different concentrations (50 ng/mL-50 μg/mL)of these ligands when bound to this cell line. Cynomolgus monkey-PD-1expressing Jurkat cells (2×10⁵) were incubated with hu-Fc ReceptorBinding Inhibitor Functional Grade (Fc-γ receptor Block at 1 μg/1×10⁶cells, Affymetrix/eBiosciense, San Diego, Calif.) for 20 minutes andblue fluorescent reactive dye, used for Live/Dead staining, was added tothe mixture (Life Technologies, San Diego, Calif.).Alexa-Fluor®-647-cynomolgus monkey PD-L1 or Alexa-Fluor®-647-cynomolgusmonkey PD-L2 were added to the cells at t 20 μg/mL immediately followedby different concentrations of mAb7, positive control 1, positivecontrol 2, or negative control antibodies (using the IgG4 backbone, in1:3 serial dilution). Cells were then allowed to incubate on ice for 1hour. After washing, cells were acquired on the BD LSRFortessa™ cellanalyzer (BD Biosciences, San Jose, Calif.). Data analyses were doneusing Flowjo software (FLOWJO LLC, Ashland, Oreg.), and geometricalmeans were calculated. IC50 values were calculated with Graph Pad Prism(Log inhibitor versus response [binding]).

Human buffy coat, purchased from Stanford Blood Center (Stanford,Calif.), was diluted with PBS and layered over Ficoll for the isolationof PBMCs. The PBMCs were washed 4 times with PBS. RBCs were lysed usingACK lysing buffer as indicated in the manufacturers protocol (LifeTechnologies, San Diego, Calif.). After the RBC lysis, cells were washedonce more and diluted in PBS at 5×10⁷ cells per mL. Half of the PBMCswere counted and frozen down in freezing media (90% FBS with 10%dimethyl sulfoxide [DMSO]) and the remaining cells were subjected toT-cell purification. From the remaining PBMCs, T lymphocytes wereisolated using a hu-specific Pan T-cell isolation kit with negativeselection as described in the manufacturer's protocol (Miltenyi Biotec,San Diego, Calif.).

For the ADCC assay, freshly-isolated T cells (target cells) werecounted, and 1×10⁶ T cells per mL were cultured in serum free X-vivo 15media (Lonza, Walkersville, Md.) and stimulated using beads coated withanti-CD3 and anti-CD28 (Dynabeads® Human T-Activator CD3/CD28 for T-CellExpansion and Activation (Life Technologies, San Diego, Calif.) and 100U/mL of recombinant-human (rh)-IL-2 (R&D Systems, Minneapolis, Minn.).Cultures were incubated at 37° C. in 5% CO2 for 72 hours. When T-cellactivation reached the 48 hour time point, PBMCs (effector cells derivedfrom the same donor) were thawed, counted, and then stimulated withrh-IL-2 (50 U/mL to 5×10⁶ cells/mL culture) for a total of 18 to 24hours in complete RPMI-1640 media with 10% FBS. On the day of the assay,both cell types were counted and tested by flow cytometry to ensureappropriate activation. For T cells, expression of high PD-1 wasexamined and for PBMCs expression level of receptors CD16, CD32, andCD64 (Fc-gamma receptor-[FcγR]III [a and b], FcγRIIa, FcγRI,respectively) that mediate ADCC was examined. Cells were plated in 5:1effector to target ratio (as this was an optimal ratio) in flat-bottom96-well tissue culture-treated plates (Fisher Scientific, Pittsburgh,Pa.) following the addition of antibodies: mAb7(IgG4 and IgG1frameworks), and positive control antibodies. All antibodies were testedat concentrations of 0.01 to 100 μg/mL (in 1:10 serial dilutions). Assaycontrols were added, including target cells alone, to evaluate maximallysis, effector cells alone, and effector to target at a 5:1 ratio withno addition of antibody. Assay plates were incubated at 37° C. in 5% CO2for 4 hours. Data were analyzed using CytoTox 96® Non-RadioactiveCytotoxicity Assay (Promega US, Madison, Wis.) as instructed by themanufacturer's protocol. The assay quantitatively measured lactatedehydrogenase (LDH), a stable cytosolic enzyme that is released uponcell lysis. Percent (%) of cytotoxicity was measured as=100×(Experimental LDH Release [OD490]/Maximum LDH Release [OD490]).Maximum LDH release was calculated when target cell alone (T cells)cells were lysed chemically to obtain maximum killing.

The ability of mAb7 to bind to hu-, cynomolgus monkey-, mouse-, andrat-PD-1 was evaluated using flow cytometry (FACS) cell-based bindingassays that included transiently transfected HEK-293T cell line, primaryactivated T cells, and Jurkat cells stably transfected with eitherhu-PD-1 or cynomolgus monkey- and mouse-PD-1. mAb7 bound to both hu-PD-1and cynomolgus monkey-PD-1 with high affinity and showed similar EC₅₀values for the 2 species (the amino acid sequence for these isoforms arethe most similar of the species analyzed). The data are summarized inTables 19, 20, and 21. Binding to mouse-PD-1 was only achieved at a highconcentration of mAb7 that is biologically irrelevant and could be dueto the affinity maturation process. No binding of mAb7 was detected forrat-PD-1 when using transfected cells or activated primary T cells(Table 19).

TABLE 19 mAb7 Binding on Primary Naïve and Activated T Cells by FACSmAb7 (% Binding) Negative Control (% Binding) Species Naïve ActivatedNaïve Activated Human T cells 9.68 ± 4.82 87.95 ± 1.65  3.49 ± 0.59 4.75± 0.59 Cynomolgus 9.21 ± 0.89 84.35 ± 0.75 5.385 ± 0.38 4.67 ± 0.47Monkey T cells Mouse T cells 4.63 ± 0.62  4.14 ± 1.87  0.93 ± 0.07 0.68± 0.10 Rat T cells 3.82 ± 1.72  0.74 ± 0.05  0.82 ± 0.16 4.36 ± 0.34

TABLE 20 mAb7 Binding to Activated Primary T Cells Obtained ExpressingPD-1 From Individual Human and Cynomolgus Monkey (EC50) Human T CellsCynomolgus Monkey T Cells Individual (pM) (pM) Individual 1 45.9  73.61Individual 2 56.11 97.07 Average ± sem 51.04 ± 5.07 85.34 ± 11.73In Table 20, each number represents EC₅₀ values for mAb7 binding to adifferent donor. The bottom row represents the average±sem. sem=Standarderror mean.

TABLE 21 mAb7 Binding to Stably-Transfected Jurkat Cell Lines with HumanPD-1 or Cynomolgus Monkey PD-1 (EC50) Jurkat/Human- Jurkat/CynomolgusmAb (IgG4) Repeat Number PD-1 (pM) Monkey-PD-1 (pM) mAb7 1 62.84 181.3 266.61 263.8 *Average ± sem 64.725 ± 1.89 222.55 ± 41.3 Positive control1 54.52 265.2 mAb1 2 57.60 267.2 *Average ± sem  56.01 ± 1.55 266.2 ±1.0 Positive control 1 181.6 380.4 mAb2 2 179.6 375.9 *Average ± sem180.6 ± 1.0 378.15 ± 2.5 In Table 21, each number represents EC₅₀ values for mAb7 binding in asingle experiment. *indicates the average±sem. mAb (IgG4)=Mononclonalantibody with IgG4 framework; PD-1=Programmed death-1; sem=Standarderror mean.

mAb7 binding to both hu- and cynomolgus monkey-PD-1 was examined usingdifferent cell based assay systems. In all systems the binding was foundto be with high affinity and specificity in both species. Using HEK-293Ttransiently-transfected cell lines that expressed either hu- orcynomolgus monkey-PD-1, mAb7 showed similar binding patterns asindicated by MFI. Minimal to no binding was observed in the parentalcell line transfected with empty vector (vehicle).

EC₅₀ values of mAb7 binding to activated hu- and cynomolgusmonkey-primary T cells were determined at 72 hours post activation, whenPD-1 expression on cell surface and cell viability were optimal (Table19). EC₅₀ values were calculated for 2 different donors of each species(Table 20). In both hu- and cynomolgus monkey-activated T cells, lowEC₅₀ values were obtained and EC₅₀ values were found to be dose betweenthe 2 species, 51.04±5.07 pM and 85.34±11.73 pM, respectively(mean±standard error mean [sem]). No binding was observed with thenegative control antibody above the baseline values.

The Jurkat T cell line, which minimally expresses hu-PD-1 receptors, wasused to generate stably-transfected hu-PD-1, cynomolgus monkey-PD-1, andmouse-PD-1 cell lines. Cell lines were sub-cloned and clones expressinghigh levels of hu-PD-1 and cynomolgus monkey-PD-1 receptors wereselected (˜400,000 PD-1 receptors per cell was the highest expressionobtained). In this system, mAb7 showed high affinity for hu-PD-1 andcynomolgus monkey-PD-1 receptors. EC₅₀ values for the two species weresimilar to the primary activated T-cell data (Table 21);EC₅₀=64.725±1.89 for hu-PD-1 and 222.55±41.3 for cynomolgus monkey-PD-1.EC₅₀ values for cynomolgus monkey-PD-1 expressing cells were morevariable than human EC₅₀ values between the 2 experimental runs. Datafor the two species were similar to data obtained with the positivecontrol antibodies used in any given repeat (Table 21). Negative controlantibody did not exhibit binding above the baseline values in any of theexperimental repeats. The ability of mAb7 to block PD-L1 and PD-L2ligands from interacting with the PD-1 receptor was examined in aPD-1-transfected Jurkat Cell line (expressing either hu-PD-1 orcynomolgus monkey-PD-1). In this assay, cells were incubated withlabeled ligands at saturating concentrations following incubation withunlabeled mAb7 at different concentrations. As shown in Table 22, mAb7inhibited PD-L1 and PD-L2 ligands from binding to the PD-1 receptor in adose-dependent manner. Positive control antibodies for PD-1 showedcomparable inhibition. The IC50 of mAb7 was comparable between hu- andcynomolgus monkey-PD-1 (880.15±289.85 and 1058±355.4 for hu-PD-L1 andhu-PD-L2, respectively; and 942.9±110.1 and 839±89.5 for cynomolgusmonkey-PD-L1 and cynomolgus monkey-PD-L2, respectively). A summary ofIC50 values is provided in Table 22. In Table 22, each number representsIC₅₀ values for mAb7 in a single experiment; * indicates theaverage±sem; ‡ indicates the repeat number in parentheses; mAbIgG4=monoclonal antibody with IgG4 framework; PD-1=Programmed death-1;PD-L1=Programmed death-ligand 1; PD-L2=Programmed death-ligand 2;sem=Standard error mean.

TABLE 22 Inhibitory Concentrations (IC₅₀; pM) for Blockade of Human PD-1or Cynomolgus Monkey-PD-1 Binding to PD-L1 and PD-L2 UsingStably-Transfected Jurkat Cell Line System Human-PD-1/Jurkat CynomolgusMonkey-PD-1/Jurkat PD-1/PD-L1 PD-1/PD-L2 PD-1/PD-L1 PD-1/PD-L2 mAb(IgG4) Blockade Blockade Blockade Blockade mAb7 (1)‡ 590.3 703.2 832.8749.5 (2)‡ 1170 1414 1053 928.5 *Average ± sem 880.15 ± 289.85  1058 ±355.4  942.9 ± 110.1   839 ± 89.5 C1 (1)‡ 1022 1226 1286 1316 (2)‡ 629811.1 861.8 1289 *Average ± sem 825.5 ± 196.5 1081.55 ± 207.45 1073.9 ±212.1 1302.5 ± 13.5 C2 (1)‡ 1715 1972 1809 1961 (2)‡ 1597 1560 1881 2111*Average ± sem 1656 ± 59  1766 ± 206 1845 ± 36  2036 ± 75

mAb7 showed weak binding for the complement component 1, q subcomponent(C1q) and CD64. C1q and CD64 (for IgG4 framework) are considered to bepotential surrogates for complement dependent cytotoxicity (CDC) andADCC, respectively. To further investigate the lack of ability of mAb7to induce the killing of T cells in vitro, an ADCC assay was performedwith activated T cells (target cells) expressing high levels of PD-1receptors and PBMCs (effector cells) that expressed high levels of Fcγreceptors. Cells were selected from 2 healthy donors. mAb7 (in itsoriginal IgG4 framework) showed minimal ADCC activity in both donors.The lack of ADCC activity was in accordance with the activity exhibitedby anti PD-1 positive control antibody in the IgG4 framework and bothwere similar to the negative control IgG4 antibody. When the anti-PD-1(mAb7 or positive control anti-PD-1 antibodies) were assessed in thehuman IgG1 framework, which is known to induce a stronger ADCC,anti-PD-1 induced ADCC up to 4-fold higher than when the antibody is inthe IgG4 framework. Maximum LDH release was calculated when target cellsalone (T cells) were chemically lysed to obtain maximum killing (killingwas estimated as 100% lysis for each donor according the assaycalculation). T cell lysis using the IgG1 framework corresponds to thelevel of PD-1 on activated T cells (donor 1 PD-1 expression as well aslysis is higher than that of donor 2), confirming the accuracy of theassay.

These results demonstrate that anti-PD-1 antibody mAb7 binds with highaffinity to hu- and cynomolgus monkey-PD-1 receptors expressed on cellswith EC₅₀ values ranging between 46 to 270 pM, depending on the testsystem. mAb7 did not bind to cells expressing mouse-PD-1 or to rat-PD-1in physiological concentrations. mAb7 blocked PD-L1 and PD-L2 ligandsfrom interacting with cell surface PD-1 receptors; IC50 values rangedfrom 500 to 1000 pM indicating its high antagonistic function inblocking PD-1 function induced via ligand binding. mAb7, in its IgG4framework, triggered minimal to no antibody-induced cytotoxicity whichis consistent with IgG4 antibody properties.

Example 12 Characterization of Anti-PD-1 Antibody mAb7 Binding to PD-1,FcRn FcγRs, and C1Q Using Label-Free Biosensors and ELISA

This example illustrates the in vitro binding affinities of mAb7 towardsrecombinant purified PD-1 from various species relevant to toxicologystudies using SPR biosensors. The ability of the Fc region of mAb7 toengage FCGRs and the FcRn was also tested by SPR to confirm that itexhibited properties consistent with those of an isotype-matchedcontrol. By ELISA, mAb7 and an isotype-matched control were assayed forbinding to human C1q. The ability of mAb7 to block the bindinginteraction of PD-1 with its ligands, PD-L1 and PD-L2, was also testedby label-free biosensors to support its mechanism of action.

All kinetic and affinity experiments were conducted at 25° C. inphosphate buffered saline (PBS)+0.01% Tween 20 running buffer, unlessstated otherwise. Kinetic studies were performed on a SPR ProteOn XPR36biosensor equipped with NLC (neutravidin-coated) chips (BioRad,Hercules, Calif.). The ProteOn was also used to determine the activeconcentrations of hu-PD-1 and cynomologus monkey-PD-1 analytes viatitration against mAb7 as the reference standard. Concentrations of theprotein analytes used here refer to “active” or “nominal” values. Theactive concentration of human Fc gamma receptor (hu-FCGR) I, humanneonatal Fc receptor (hu-FcRn) (Lot No R3091), and cynomolgusmonkey-neonatal Fc receptor (cynomolgus monkey-FcRn) (Lot No JCR) weredetermined using calibration-free concentration analysis (CFCA)experiments on a Biacore T200 equipped with CM5 sensor chips (GE LifeSciences, Marlborough, Mass.). All other analytes were used at theirnominal concentrations as determined by light absorbance at A280 nm withan appropriate extinction coefficient. Solution affinities weredetermined at room temperature (approximately 23° C.) using a KinExAinstrument 3000 or 3200 equipped with autosampler (Sapidyne). Secondarydetection antibodies were labeled with DyLight 650 (PierceBiotechnology, Grand Island, N.Y.) according to the manufacturer'sinstructions. Immunoglobulin G (IgG) biotinylations were performed at anequimolar ratio of linker:IgG using EZ-Link™ Sulfo-NHS-LC-LC-Biotin(Pierce Biotechnology Grand Island, N.Y.) according to themanufacturer's instructions. Unless stated otherwise, immobilized IgGswere regenerated with a “Pierce/salt” cocktail comprising a 2:1 v/vmixture of Pierce IgG elution buffer (pH 2.8)/4 M sodium chloride(NaCl).

The binding interactions of hu-PD-1 and cynomolgus monkey-PD-1 (bothHis-tagged monomers) towards mAb7 were determined in solution using theKinExA method in a running buffer of PBS+0.01% Tween 20 and a samplebuffer of PBS+0.01% Tween 20+1 μg/I bovine serum albumin (BSA). Twodifferent assay formats were employed. In the first assay format, mAb7was titrated into a constant concentration of hu-PD-1 (nominal 200, 400,or 4000 pM) and samples were allowed to reach equilibrium. Free hu-PD-1was captured on polymethylmethacrylate (PMMA) beads that had been coated(by adsorption) with anti-hu-PD-1 monoclonal antibody mAb7 (preparedin-house under non-Good Laboratory Practices (GLP) conditions [Lot NoR5432]). Bead-captured hu-PD-1 was then detected with 0.5 μg/mLDylight-labeled anti-His mAb (R&D Systems, Minneapolis, Minn.). In thesecond assay format, hu-PD-1 or cynomolgus monkey-PD-1 were titratedinto a constant concentration of mAb7 (20, 50, 100, or 500 pM) and thesemixtures were allowed to equilibrate. Free mAb7 was captured on PMMAbeads that had been adsorbed with a blocker anti-idiotypic mouseanti-mAb7 mAb 1699.1H6 that binds specifically to free mAb7 but notPD-1-saturated mAb7. Bead-captured mAb7 was then detected withDylight-labeled goat anti-hu-IgG (H+L) (Jackson ImmunoResearch Inc, WestGrove, Pa.). All titrations were prepared as a 12-membered 2-folddilution series varying the top nominal binding site concentration tofall within the range 1 nM to 10 nM, depending on the experiment.Samples were allowed to equilibrate for up to 48 hours and injectionvolumes were adjusted per experiment to give a total signal that fellwithin the range 0.7 V to 1.9 V. All samples were injected in duplicatecycles. The data from up to 4 independent experiments per interactionwere analyzed globally using the N-curve tool in the analysis softwareand fit to a simple bimolecular model where mAb7's concentration wasused as the reference concentration. The global analysis reports thebest fit values (and 95% confidence interval) for the K_(D) and theapparent active binding site concentration of PD-1.

The binding affinities of hu-PD-1 and cynomolgus monkey-PD-1 towardsmAb7 in solution were indistinguishable from one another when studiedusing a KinExA assay; the apparent equilibrium dissociation constant(K_(D)) values at 23° C. were determined to be 17 pM or 23 pM forhu-PD-1 (when studied in opposing assay orientations) and 28 pM forcynomolgus monkey-PD-1; these three values werestatistically-indistinguishable from one another. These values wererecapitulated by surface plasmon resonance (SPR) biosensor measurements,which yielded K_(D) values of 42 pM for hu-PD-1 and 69 pM for cynomolgusmonkey-PD-1 at 25° C., and 109 pM for hu-PD-1 and 115 pM for cynomolgusmonkey-PD-1 at 37° C. mAb7 bound mouse-PD-1 with a K_(D) value of 0.9 μMand no binding was detected towards rat-PD-1 when tested at 0.5 μM withSPR kinetic analysis. Label-free biosensors were used to demonstratethat mAb7 blocks hu-PD-1 from binding to its natural ligands, humanprogrammed death-ligand 1 (hu-PD-L1) and human programmed death-ligand 2(hu-PD-L2). SPR was further used to confirm that mAb7 bound a range offragment crystallizable (Fc) gamma receptors (FCGR) and the neonatal Fcreceptor (FcRn) from both human and cynomolgus monkey species with thesame kinetics and specificity as an isotype-matched control. By ELISA,mAb7 showed negligible binding to complement component 1, q subcomponent(C1q), consistent with the behavior of an isotype-matched control.Overall, mAb7 binds both hu-PD-1 and cynomolgus monkey-PD-1 with highbinding affinity and specificity but has low affinity towards mouse-PD-1and negligible binding towards rat-PD-1.

Table 23 summarizes the kinetic and affinity data obtained for theinteraction analysis of mAb7 with PD-1 from various species. mAb7 boundhu-PD-1 and cynomolgus monkey-PD-1 with statistically indistinguishableapparent affinities when these interactions were measured in solution at23° C. using the KinExA method; apparent K_(D) values were 17 pM and 23pM for hu-PD-1 (when studied in two opposing assay orientations) and 28pM for cynomolgus monkey-PD-1. The KinExA values were corroborated bykinetic measurements performed by SPR, which gave apparent K_(D) valuesat 25° C. of 42±11 pM (n=10) for hu-PD-1 and 69±24 pM (n=10) forcynomolgus monkey-PD-1. SPR measurements at 37° C. showed 2-fold weakeraffinities than those at 25° C.; K_(D) values at 37° C. were determinedto be 109±8 pM (n=15) for hu-PD-1 and 115±15 pM (n=8) for cynomolgusmonkey-PD-1. mAb7 bound mouse PD-1 with an apparent K_(D) of 0.9±0.1 pM(n=5) when measured at 25° C. by SPR, corresponding to a 20,000-foldweaker affinity than that for hu-PD-1. No binding was detected forrat-PD-1 when tested at 0.5 μM at 25° C. by SPR, although the rat-PD-1proteins were confirmed active via their clear binding to positivecontrols, mouse-PD-L1 and mouse-PD-L2. The K_(D) values for the KinExAmeasurements represent the best fit and 95% confidence interval of aglobal analysis of N independent experiments, while those for the SPRmeasurements represent the mean±standard deviation of n independentexperiments on a ProteOn NLC sensor chip. In Table 23, h or hu=Human;k_(a)=Association rate constant; k_(d)=Dissociation rate constant;K_(D)=Equilibrium dissociation constant; KinExA=Kinetic exclusion assay;mAb=Monoclonal antibody; Ms=Molar per second; n=Number; ND=Notdetermined; PD-1=Programmed death-1; s=Second; SPR=Surface plasmonresonance; Temp=Temperature; *mAb7's interaction with hu-PD-1 wasstudied in two opposing assay orientations as described in Bee et al,2012.

TABLE 23 Summary of Kinetic and Affinity Constants Obtained for theInteractions of PF 06801591 with Human PD-1, Cynomologus Monkey PD-1,and Mouse PD-1 Temp (° C.) Method PD-1 k_(a) (1/Ms) k_(d) (1/s) K_(D)(pM) n 23 KinExA Human ND ND 23 (28-18) 3 (Titrate mAb)* 23 KinExA HumanND ND 17 (24-12) 3 (Titrate PD-1)* 25 SPR Human (4.2 ± 0.6) × 10⁵  (1.8± 0.4) × 10⁻⁵ 42 ± 11 10 37 SPR Human (8.4 ± 2.0) × 10⁵  (8.5 ± 1.2) ×10⁻⁵ 109 ± 28  15 23 KinExA Cynomologus ND ND 28 (34-23) 4 Monkey(Titrate PD-1) 25 SPR Cynomologus (4.3 ± 1.4) × 10⁵  (2.9 ± 0.4) × 10⁻⁵69 ± 24 10 Monkey 37 SPR Cynomologus (9.8 ± 1.1) × 10⁵ (1.12 ± 0.07) ×10⁻⁴ 115 ± 15  8 Monkey 25 SPR Mouse (6.0 ± 0.5) × 10³  (5.2 ± 0.4) ×10⁻³ (9.0 ± 1.0) × 5 10⁵

Table 24 shows that binding kinetics and specificity for mAb7 over arange of Fc receptors, including FCGRs and FcRn from both human andcynomolgus monkey recapitulated those values expected for anisotype-matched isotype control, hu-IgG4. For example, mAb7 boundhu-FCGRI with an apparent K_(D) value (mean±standard deviation) at 25°C. of 146±42 pM (n=14) compared with 177±71 pM (n=4) for the isotypecontrol. All other interactions were characterized by weak affinities(K_(D) values˜1 μM or higher). Kinetic analysis was performed on aProteOn NLC chip at pH 7.4 for FCGR and pH 5.9 for FcRn. In Table 24,CD=Cluster of differentiation; cy=Cynomologus monkey; FCGR=Fc gammareceptor; FcRn=Neonatal Fc receptor; hu=Human; k_(a)=Association rateconstant; k_(d)=Dissociation rate constant; K_(D)=Equilibriumdissociation constant; Ms=Molar per second; n=Number; s=Second.

TABLE 24 Analyte mAb7 Isotype-matched Control hu-FCGRI (hu-CD64) k_(a) =(9.1 ± 1.7) × 10⁶ (1/Ms) k_(a) = (1.0 ± 0.3) × 10⁶ (1/Ms) k_(d) = (1.3 ±0.3) × 10⁻³ (1/s) k_(d) = (1.8 ± 0.4) × 10⁻³ (1/s) K_(D) = 146 ± 42 pM(n = 14) K_(D) = 177 ± 71 pM (n = 4) hu-FCGRIIa (hu-CD32a) 131H  3.6 μM3.9 μM hu-FCGRIIa (hu-CD32a) 131R  0.8 μM 0.4 μM hu-FCGRIIb (hu-CD32b) 1.1 μM 0.7 μM hu-FCGRIIIa (hu-CD16a) 158F Barely binds at 5 μM Barelybinds at 5 μM hu-FCGRIIIa (hu-CD16a) 158V Barely binds at 1 μM Barelybinds at 1 μM hu-FcRn 0.93 μM 0.82 ± 0.06 μM (n = 3) CynomolgusMonkey-FCGRIIa  2.4 μM  2.6 ± 0.5 μM (n = 3) Cynomolgus Monkey-FCGRIIIa 2.7 μM  3.2 ± 0.1 μM (n = 3) 42R Cynomolgus Monkey-FCGRIIIa  1.6 μM 1.9 ± 0.2 μM (n = 3) 42S Cynomolgus Monkey-FcRn 0.61 μM 0.57 ± 0.04 μM(n = 3)

In addition, mAb7 barely bound C1q by ELISA, consistent with thebehavior of the isotype-matched control, hu-IgG4 (data not shown). Itsbinding was even lower than the low binding of human immunoglobulin G2(hu-IgG2). In contrast, the positive controls, hu-IgG1 and humanimmunoglobulin G3 (hu-IgG3), gave high binding signals. Using both SPRand Octet biosensors and different assay formats (both premix andclassical sandwich assay formats), it was demonstrated that mAb7 blockedhu-PD-1 binding to its natural ligands, hu-PD-L1 and hu-PD-L2.

These data demonstrate that that mAb7 binds with high affinity and highspecificity towards hu-PD-1. When measured in solution at 23° C. usingthe KinExA method, mAb7 bound hu-PD-1 and cynomolgus monkey-PD-1 withstatistically-indistinguishable apparent K_(D) values of 17 pM and 23 pMfor hu-PD-1 (when studied in alternate assay orientations) and 28 pM forcynomolgus monkey-PD-1i. mAb7 showed a 20,000-fold weaker affinity tomouse-PD-1 (apparent K_(D) of 0.9 μM at 25° C. by SPR) and no detectablebinding to rat-PD-1 when tested at 0.5 μM. mAb7 and an isotype-matchedcontrol hu-IgG4 bound with similar kinetics and specificity when testedagainst a panel of hu- and cynomolgus monkey-Fc receptors, when measuredat 25° C. by SPR. mAb7 bound hu-C1q with negligible signal when testedby ELISA, consistent with hu-IgG4 isotype controls, and its binding waseven lower than that of hu-IgG2. It was demonstrated that mAb7 blockshu-PD-1 from binding to its natural ligands, hu-PD-L1 and hu-PD-L2.

Example 13 Combination Treatment with Anti-PD-1 Antibody and Anti-OX40Antibody

This example illustrates the effects of treatment with anti-PD-1antibody in combination with anti-OX40 antibody in a mouse model forcolon cancer.

For this study, C57B6/J female mice (6-8 weeks old) were inoculatedsubcutaneously with the MC-38 murine colon tumour (a grade IIIadenocarcinomat) at 5×10⁵ cells/mouse. At Day 10, mice were randomizedand treatment began at 10 mg/kg anti-mouse-PD-1 and 1 mg/kganti-mouse-OX-40. Treatments were administered i.p. at 15 days 10, 12,and 14 for anti-OX40 antibody (1 mg/kg); and at days 10, 12, 14, 17, 23,and 25 for anti-PD-1 antibody (10 mg/kg). Tumor volume was measuredtwice a week. The study was terminated at day 34. Statistical analyseswere performed using 2-way Anova to compare the averages between eachtwo groups at a single time point. The results are summarized in Table25. Vin table 25, values indicate tumor volume at the indicated dayaverage of 8 mice per group±(standard error mean) sem; N=number pergroup, mm²=cubic millimeter. The isotype control included theappropriate isotype of each antibody at 1 mg/kg for anti-OX40 and 10mg/kg for anti-PD-1.

TABLE 25 Tumor volume in MC38 bearing mice after treatment withanti-PD-1 antibody and anti-OX40 antibody Treatment Tumor volume (mm²) %of Tumor Groups Day 10 Day 24 Day 27 Day 34 N free mice Isotype control67. ± 7.9 832. ± 139.0 1408 ± 252.4  1866 ± 279.0 8   0% Anti-PD-1  67 ±8.0  312 ± 63.2   540 ± 125.0   939 ± 169.0 8   0% Anti-OX40  67 ± 7.5 302 ± 64.1  370 ± 77.2   720 ± 145.0 8   0% Anti-PD-1 +  67 ± 7.4  190± 38.0  255 ± 51.2 423.0 ± 90.0  8 12.5% Anti-OX40

Beginning at Day 24, all anti-PD-1 antibody and/or anti-OX-40 antibodytreated groups showed significantly smaller tumor volumes when comparedto the control group (Table 25). At Day 24, the combination (anti-PD-1antibody plus anti-OX40 antibody) treated group showed significance ofp≦0.0001 when compared to the isotype control group, while each singleantibody treatment showed significance of p≦0.01 when compared to thecontrol group. At Day 34, all treated groups showed significance ofp≦0.0001 when compared to the control group. At Day 34 the combinationtreated grouped showed differences when compared to either anti-PD-1antibody alone treated group or anti OX-40 antibody alone treated group.These results demonstrate that treatment with both anti-PD-1 antibodyand anti-OX40 antibody slowed tumor growth when compared to treatmentwith either antibody alone.

Example 14 Effect of Vaccine on Expression of PD-1 on Activated T Cells

This example illustrates the effect of a DNA-based vaccine expressing ahuman Prostate Specific Membrane Antigen (PSMA) on the expression ofPD-1 on CD3⁺CD4 (Table 26) and CD3⁺CD8 T cells (Table 27) in thepresence of anti-CTLA4 monoclonal antibody tremelimumab (a checkpointinhibitor).

In Vivo Study Procedures.

Three groups of Chinese cynomolgus macaques (n=8/group) were used in thestudy. Animals in Groups 1 and 2 were intramuscularly injected with avaccine that contains an AdC68 adenovirus vector (SEQ ID NO:44) encodingamino acids 15-750 of the human PSMA (at a total dose of 2e11 VP) whileanimals in Group 3 received no vaccination. The complete sequence of theAdC68 adenovirus vector is provided in SEQ ID NO:44. In addition,immediately following vaccination animals in Group 2 were treated with50 mg anti-CTLA4 monoclonal antibody tremelimumab by subcutaneousinjections. Whole blood samples from each animal before (Pre) and aftervaccination were collected at Days 9, 15 and 29 and analyzed for thefrequency of PD-1 expressing CD3+CD4 and CD3′CD8 T cells by flowcytometry.

CD3+ CD4 and CD8 T Cell Phenotyping.

Leukocyte populations were phenotyped by performing multi-color flowcytometry analysis on freshly collected whole blood. Briefly, wholeblood was stained for 20 min at room temperature with an antibodycocktail containing CD3-V500 (done SP34-2), CD4-BV605 (done L200),CD8-APC.H7 (Clone SK1), PD1-AF488 (clone EH12.2H7, Biolegend),PDL1-BV421 (done MIH1), Lag3-PE.Cy7 (done 11E3, Novus), CD69-PE.TexasRed (clone TP1.553, Beckman Coulter), TIM3-APC (clone 344823, R&DSystems), CD20-PE.Cy5.5 (clone 2H7, eBioscience), CD14-AF700 (doneM5E2), CD16-PE.Cy5 (done 3G8), CD56-PerCP.Cy5.5 (done B159), andCD11c-PE (done SHCL3). All antibodies were purchased from BD unlessotherwise indicated. The samples were treated with RBC lysis buffer(BD), washed, mixed with 50 ml of liquid counting beads (BD) andacquired on LSR Fortessa (BD). The data were analyzed using FlowJo (TreeStar Inc, USA). Results of PD-1 expression on CD3⁺CD4 T cells arepresented in Table 26 and results of PD-1 expression on CD3⁺CD8 T cellsare presented in Table 27. The induction of PD-1 was observed 9 daysafter vaccination and resolved to levels seen in naive animals (Group 3)by day 29 post vaccination. These results indicate that the vaccinevector given together with anti-CTLA4 antibody can elicit PD-1expression on CD3⁺ CD4 and CD8 T cells in nonhuman primates.

TABLE 26 PD-1 Expression Kinetics on CD3⁺CD4 T Cells in CynomolgusMacaques Time Individual % PD-1⁺ CD3⁺CD4 T cells in total CD3⁺CD4 Tcells point Group 1 2 3 4 5 6 7 8 Pre 1 16.0 7.473 8.275 12.441 12.93512.481 10.93 12.649 2 9.757 11.383 7.795 5.943 13.279 24.959 12.11210.375 3 11.577 7.577 9.943 Day 9 1 18.124 11.72 11.977 18.861 20.65710.623 11.766 12.792 2 32.07 29.001 38.593 27.516 25.962 34.568 27.43528.378 3 18.608 10.101 13.205 Day 15 1 14.87 10.302 8.27 15.742 13.6098.763 10.407 9.82 2 16.421 14.578 18.138 15.667 20.927 27.778 16.61217.583 3 16.878 10.607 10.878 Day 29 1 14.819 9.39 8.412 14.051 14.4369.576 10.524 11.116 2 11.594 10.209 12.971 8.293 20.989 26.388 14.12613.868 3 14.182 9.098 14.138

TABLE 27 PD-1 Expression Kinetics on CD3⁺CD8 T Cells in CynomolgusMacaques Time Individual % PD-1⁺ CD3⁺CD8 T cells in total CD3⁺CD8 Tcells point Group 1 2 3 4 5 6 7 8 Pre 1 10.8 4.9 10.1 5.7 11.9 13.5 7.19.6 2 5.3 19.4 5.0 20.2 16.9 29.8 7.7 8.7 3 8.8 6.9 4.3 Day 9 1 5.3 7.012.3 8.1 16.1 13.2 5.8 11.3 2 15.6 22.5 7.4 29.5 20.7 26.2 11.41 10.2 311.5 5.8 5.7 Day 15 1 7.4 6.9 11.7 8.2 16.2 15.4 6.8 14.0 2 7.6 21.610.8 21.8 27.1 25.7 14.2 10.5 3 12.3 7.4 6.4 Day 29 1 9.4 7.5 15.3 9.814.7 16.7 5.9 14.7 2 8.5 20.3 8.0 22.5 26.0 22.4 9.1 9.9 3 11.6 8.6 8.5

Example 15 Effect of Anti-PD-1 Antibody on Antigen-Specific T-CellResponse Induced by a Vaccine Expressing Human PSMA

This example illustrates the effect of anti-PD1 antibodies on the IFNγCD4 and CD8 T-cell responses induced by a vaccine containing an AdC68adenovirus vector that expresses human PSMA in cynomolgus macaques.

Intracellular Cytokine Staining (ICS) Assay.

Briefly, PBMCs from individual animals were co-incubated with pools ofpeptides from a 15mer peptide library overlapping by 11 amino acidsspanning the entire sequence of the respective antigen, each peptide at2 μg/ml. The plates were incubated ˜16 hours at 37° C., 5% CO₂. Thecells were then stained to detect intracellular IFNγ expression fromCD8⁺ T cells and fixed. Cells were acquired on a flow cytometer. Thefrequency of response was normalized to the number of IFNγ CD8⁺ T cellsper million CD8⁺ T cells, with the responses in dimethyl sulfoxidecontrol wells, which contained no peptide, subtracted. The antigenspecific responses in the tables represent the sum of the responses tothe corresponding antigen specific peptide pools.

In Vivo Study Procedures.

Briefly, Five groups of Chinese cynomolgus macaques (n=8/group) wereintramuscularly injected with a vaccine containing an AdC68 adenovirus(SEQ ID NO:44) that encodes amino acid 15-750 of human PSMA (hPSMA) attotal dose of 2e11 VP. The vaccination in Groups 2, 4 and 5 wasimmediately followed by subcutaneous injections of 50 mg anti-CTLA4monoclonal antibody tremelimumab and/or anti-PD-1 monoclonal antibodymAb7 given subcutaneously (groups 3 and 4) or intravenously (group 5) at10 mg/kg. PBMCs were isolated from each animal and subjected to an ICCSassay to measure human PSMA specific IFN□ CD4 (Day 9 or day 29) or CD8T-cell responses (Day 29). The amino acid sequence of the full lengthhuman PSAM is provided in SEQ ID NO:42. The sequence of the AdC68adenovirus vector expressing amino acids 15-750 of the human PSMA isprovided in SEQ ID NO:44.

Results.

Results are presented in Tables 29, 30, and 31. The data demonstratedthat both anti-CTLA4 and anti-PD-1 individually can increase thefrequency of IFNγ CD4 and CD8 T-cell responses induced by the vaccine.

TABLE 28 Peptide sequence information for peptide pools used in theELISpot and ICCS assays to induce antigen-specific IFNγ T-cellresponses. Antigen Peptide library pools Human 185 sequential 15merpeptides, overlapping by 11 amino acids, PSMA covering the entire fulllength hPSMA protein sequence (amino acid sequence: 1-750) assayed asthree separate pools.

TABLE 29 IFNγ CD8 T-cell Responses Induced by the vaccine on Day 9Individual hPSMA specific IFNγ CD8 T-cell titer Checkpoint MAB7(IFNγ⁺CD8 cells/1e6 CD8 T cells, background adjusted) Geo Groupinhibitor(s) route 1 2 3 4 5 8 7 8 mean 1 None n/a 1072 1 1453 6722 1 11055 503 92.91 2 tremelimumab n/a 7278 356 423 6009 954 15862 935 25111977 3 mAb7 SC 2077 6537 3138 449 1302 4063 769 487 1575 4 tremelimumabSC 222 667 1199 6879 933 1959 40349 6901 2235 mAb7 5 tremelimumab IV1037 1735 2371 489 7435 1 20947 4613 1052 mAb7

TABLE 30 IFNγ CD4 T-cell Responses Induced by the Vaccine on Day 9Individual hPSMA specific lFNγ CD4 T-cell titer Checkpoint mAb7(IFNγ⁺CD4 cells/1e6 CD4 T cells, background adjusted) Geo Groupinhibitor(s) route 1 2 3 4 5 6 7 8 mean 1 None n/a 1 1 1 1 1 1 1 1 1 2tremelimumab n/a 665 259 1 11914 271 326 1 1747 154 3 mAb7 SC 174 669457 193 194 1 192 159 125 4 tremelimumab + SC 1955 1 435 1764 736 13071852 471 401 mAb7 5 tremelimumab + IV 858 1567 1398 1102 163 705 49381029 1024 mAb7

TABLE 31 IFNγ CD4 T-cell Responses Induced by the Vaccine on Day 29Individual hPSMA specific IFNγ CD4 T-cell titer Checkpoint mAb7(lFNγ⁺CD4 cells/1e6 CD4 T cells, background adjusted) Geo Groupinhibitor(s) route 1 2 3 4 5 6 7 8 mean 1 None n/a 1 506 246 143 504 1 11 24 2 tremelimumab n/a 175 63 1021 2845 867 662 265 372 230 3 mAb7 SC129 1 145 1 230 1 97 1 12 4 tremelimumab + SC 816 107 527 315 168 10841131 135 377 mAb7 5 tremelimumab + IV 332 1496 559 360 221 361 1164 248468 mAb7

Example 16 Effect of Anti-PD-1 Antibodies on IFNγ T-Cell ResponsesInduced by a Vaccine Co-Expressing a Human Prostate Specific MembraneAntigen (PSMA), Prostate Stem Cell Antigen (PSCA), and Prostate SpecificAntigen (PSA)

In Vivo Study Procedures. Briefly, three groups of Chinese cynomolgusmacaques (n=4/group) were used in the study. Animals in Group 1 wereintramuscularly injected with vehicle. Animals in Group 2 wereadministered the anti-PD-1 monoclonal antibody mAb7 subcutaneously at 20mg/kg. Animals in Group 3 were treated with a vaccine containing anAdC68 adenovirus vector co-expressing three human prostate associatedantigens (PSMA, PSCA and PSA) (Vector AdC68W-734), at a total dose of6e11 VP, immediately followed by subcutaneous injections of 150 mganti-CTLA4 antibody tremelimumab and anti-PD-1 monoclonal antibody mAb7at 20 mg/kg. Twenty eight days after the injections, animals wereboosted with vehicle (group 1), anti-PD-1 monoclonal antibody mAb7subcutaneously at 20 mg/kg (group 2) or a DNA plasmid (Plasmid 458)expressing three human prostate cancer antigens (PSMA, PSCA and PSA)delivered by electroporation immediately followed by subcutaneousinjections of 150 mg anti-CTLA4 and anti-PD-1 monoclonal antibody at 20mg/kg (group 3). Forty three days after the prime first injections,PBMCs were isolated from each animal and subjected to an ELISPOT assays,to measure PSMA, PSCA, and PSA specific, IFNγ T-cell responses.

The complete sequence of Vector AdC68W-734 used in the study is providedin SEQ ID NO:45 of the present disclosure and in SEQ ID NO:63 ofInternational Application Publication WO2015/063647. The construction ofVector AdC68W-734 is also described in WO2015/063647, the disclosure ofwhich is incorporated herein by reference. The AdC68W-734 vector andPlasmid 458 each comprises (1) a nucleotide sequence encoding aminoacids 15-750 of human PSMA having the sequence of SEQ ID NO:42, (2) anucleotide sequence encoding amino acids 25-261 of human PSA having thesequence of SEQ ID NO:47, and (3) a nucleotide sequence encoding thefull length human PSCA of SEQ ID NO:48.

IFNγ ELISPOT Assay.

Briefly, peripheral blood mononuclear cells (PBMCs) from individualanimals were co-incubated in duplicate with pools of peptides from a15mer peptide library overlapping by 11 amino acids spanning the entiresequence of the respective antigen, each peptide at 2 μg/ml. The plateswere incubated for ˜16 hours at 37° C., 5% CO₂, then washed anddeveloped, as per manufacturer's instruction. The number of IFNγ spotforming cells (SFC) was counted with a CTL reader. The average of theduplicates was calculated and the response of the negative controlwells, which contained no peptides, subtracted. The SFC counts were thennormalized to describe the response per 1e6 PBMCs. The antigen specificresponses in the tables represent the sum of the responses to thecorresponding antigen specific peptide pools.

Results.

The ELISpot results, which are presented in Table 33, demonstrate thatthe animals that received the vaccine with subcutaneous anti-PD-1antibody and anti-CTLA4 antibody exhibited a robust increase in IFNγT-cell response to at least one prostate cancer antigen, while therewere no IFNγ T-cell responses to these antigens in the vehicle group(Group 1) or the group administered anti-PD-1 antibody alone (Group 2).

TABLE 32 Peptide sequence information for peptide pools used in theELISpot and ICCS assays to induce antigen-specific IFNγ T-cell responsesAntigen Peptide library pools PSMA 185 sequential 15mer peptides,overlapping by 11 amino acids, covering the entire full length PSMAprotein sequence (amino acid sequence: 1-750) assayed as three separatepools. PSCA 28 sequential 15mer peptides, overlapping by 11 amino acids,covering the entire full length PSCA protein sequence (amino acidsequence: 1-123) assayed as a single separate pool. PSA 62 sequential15mer peptides, overlapping by 11 amino acids, covering the entire fulllength PSA protein sequence (amino acid sequence: 1-246) assayed as onesingle separate pool.

TABLE 33 Effect of Anti-PD-1 antibody on antigen-specific IFNγ ELISpotT-cell responses induced by vaccine in cynomolgus macaques. CheckpointStimulating Individual IFNγ ELISpot T-cell titer Group Vaccineinhibitor(s) antigen (SFC/1e6 PBMCs, background subtracted) 1 n/a n/aPSMA 2   2 2 4 2 n/a mAb7 10   1 7 3 3 + tremelimumab + 1360 1263 24413224 mAb7 1 n/a n/a PSCA 0   0 1 1 2 n/a mAb7 0   0 4 4 3 +tremelimumab + 91  18 488 416 mAb7 1 n/a n/a PSA 0   0 5 2 2 n/a mAb7 0  0 3 0 3 + tremelimumab + 404 [1267] 527 512 mAb7 Values in bracketrepresent the responses where at least one assay replicate was above theupper limit of quantification for the assay (1333 SFC/1e6 PBMC). Valuesof 0 SFC/1e6 PBMC were converted to 1 for calculation of Geometric mean.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

It is claimed:
 1. An isolated antagonist antibody that specificallybinds to PD-1 and comprises: a heavy chain variable region (VH)comprising a VH complementarity determining region one (CDR1), VH CDR2,and VH CDR3 of the VH having an amino acid sequence selected group thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 5; and SEQ IDNO: 6; and a light chain variable region (VL) comprising a VL CDR1, VLCDR2, and VL CDR3 of the VL having an amino acid sequence selected fromthe group consisting of SEQ ID NO: 2; SEQ ID NO:7; SEQ ID NO: 8; and SEQID NO:
 9. 2. The isolated antagonist antibody of claim 1, wherein theantibody comprises a VH CDR1 comprising the amino acid sequence of SEQID NO: 13, 14, or 15, a VH CDR2 comprising the amino acid sequence ofSEQ ID NO: 16, 17, 24, 25, 27, 28, 35, or 36, a VH CDR3 comprising theamino acid sequence shown in SEQ ID NO: 18, 23, 26, or 37, a VL CDR1comprising the amino acid sequence shown in SEQ ID NO:10, 22, 30, or 32,a VL CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11, 20,or 33 and a VL CDR3 comprising the amino acid sequence shown in SEQ IDNO: 12, 21, 31, or
 34. 3. The isolated antagonist antibody of claim 1,wherein the antibody comprises a VH comprising the amino acid sequenceshown in SEQ ID NO: 3, 4, 5, or 6, or a variant thereof with one orseveral conservative amino acid substitutions in residues that are notwithin a CDR.
 4. The isolated antagonist antibody of claim 3, whereinthe antibody comprises a VL comprising the amino acid sequence shown inSEQ ID NO: 2, 7, 8, or 9, or a variant thereof with one or several aminoacid substitutions in amino acids that are not within a CDR.
 5. Theisolated antagonist antibody of claim 1, wherein the antibody comprisesa VH comprising the amino acid sequence shown in SEQ ID NO: 3, 4, 5, or6, and a VL comprising the amino acid sequence shown in SEQ ID NO: 2, 7,8, or
 9. 6. The isolated antagonist antibody of claim 1, wherein theantibody comprises a heavy chain comprising the amino acid sequenceshown in SEQ ID NO: 29 or 38 and a light chain comprising the amino acidsequence shown in SEQ ID NO:
 39. 7. The isolated antagonist antibody ofclaim 1, wherein the antibody comprises a constant region.
 8. Theisolated antagonist antibody of claim 7, wherein the antibody has anisotype that is selected from the group consisting of IgG₂, IgG_(2Δa),IgG₄, IgG_(4Δb), IgG_(4Δc), IgG₄ S228P, IgG_(4Δb) S228P and IgG_(4Δc)S228P.
 9. The isolated antagonist antibody of claim 7, wherein theconstant region is IgG₄ S228P.
 10. The isolated antagonist antibody ofclaim 1, wherein each CDR of the antibody is defined in accordance withthe Kabat definition, the Chothia definition, the combination of theKabat definition and the Chothia definition, the AbM definition, or thecontact definition of CDR.
 11. An isolated antagonist antibody thatspecifically binds to PD-1 and competes for binding to PD-1 with, and/orbinds to an epitope that is the same as or overlaps with the epitope onPD-1 recognized by, the antibody of claim
 1. 12. An isolated anti-PD-1antibody, wherein the antibody comprises a VH CDR1 comprising the aminoacid sequence of SEQ ID NO: 13, a VH CDR2 comprising the amino acidsequence of SEQ ID NO: 17, a VH CDR3 comprising the amino acid sequenceshown in SEQ ID NO: 23, a VL CDR1 comprising the amino acid sequenceshown in SEQ ID NO: 10, a VL CDR2 comprising the amino acid sequenceshown in SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequenceshown in SEQ ID NO:
 21. 13. The isolated antibody of claim 1, whereinthe antibody promotes IFNγ and/or TNF secretion from T cells.
 14. Theisolated antibody of claim 1, wherein the antibody promotesproliferation of T cells.
 15. The isolated antibody of claim 1, whereinthe antibody inhibits tumor growth.
 16. The isolated antibody of claim1, wherein the antibody binds human PD-1 and mouse PD-1.
 17. Theisolated antibody of claim 16, wherein the antibody binds human PD-1with an affinity of about 0.73 nM at 25° C. as measured by surfaceplasmon resonance.
 18. An isolated cell line that produces the antibodyof claim
 1. 19. An isolated nucleic acid encoding the antibody ofclaim
 1. 20. A recombinant expression vector comprising the nucleic acidof claim
 19. 21. A host cell comprising the expression vector of claim20.
 22. A hybridoma capable of producing the antibody of claim
 1. 23. Amethod of producing an anti-PD-1 antagonist antibody, the methodcomprising: culturing a cell line that recombinantly produces theantibody of claim 1 under conditions wherein the antibody is produced;and recovering the antibody.
 24. A method of producing an anti-PD-1antagonist antibody, the method comprising: culturing a cell linecomprising nucleic acid encoding an antibody comprising a heavy chaincomprising the amino acid sequence shown in SEQ ID NO: 29 or 38 and alight chain comprising the amino acid sequence shown in SEQ ID NO: 39under conditions wherein the antibody is produced; and recovering theantibody.
 25. The method of claim 21, wherein the heavy and light chainsof the antibody are encoded on separate vectors.
 26. The method of claim21, wherein the heavy and light chains of the antibody are encoded onthe same vector.
 27. A pharmaceutical composition comprising theantibody of claim 1, and a pharmaceutically acceptable carrier.
 28. Akit for the treatment of cancer comprising the pharmaceuticalcomposition of claim
 27. 29. A method for treating cancer in a subjectin need thereof, the method comprising administering to the individualan effective amount of the anti-PD-1 antibody of claim 1 such that oneor more symptoms associated with the cancer is ameliorated in theindividual.
 30. The method of claim 29, wherein the cancer is selectedfrom the group consisting of gastric cancer, sarcoma, lymphoma,Hodgkin's lymphoma, leukemia, head and neck cancer, squamous cell headand neck cancer, thymic cancer, epithelial cancer, salivary cancer,liver cancer, stomach cancer, thyroid cancer, lung cancer, ovariancancer, breast cancer, prostate cancer, esophageal cancer, pancreaticcancer, glioma, leukemia, multiple myeloma, renal cell carcinoma,bladder cancer, cervical cancer, choriocarcinoma, colon cancer, oralcancer, skin cancer, and melanoma.
 31. The method of claim 29 whereinthe individual is a previously treated adult patient with locallyadvanced or metastatic melanoma, squamous cell head and neck cancer(SCHNC), ovarian carcinoma, sarcoma, or relapsed or refractory classicHodgkin's Lymphoma (cHL).
 32. The method of claim 29, wherein theanti-PD-1 antibody is administered at a dosage of about 0.5 mg/kg, about1.0 mg/kg, about 3.0 mg/kg, or about 10 mg/kg.
 33. The method of claim29, wherein the anti-PD-1 antibody is administered once every 7, 14, 21,or 28 days.
 34. The method of claim 29, wherein the anti-PD-1 antibodyis administered intravenously or subcutaneously.
 35. The method of claim29, wherein the method further comprises administering an effectiveamount of a second therapeutic agent.
 36. The method of claim 35,wherein the second therapeutic agent is selected from the groupconsisting of an anti-CTLA4 antibody, an anti-4-1 BB antibody, a secondPD-1 antagonist, an anti-PD-L1 antibody, an anti-TIM3 antibody, ananti-LAG3 antibody, an anti-TIGIT antibody, an anti-OX40 antibody, ananti-GITR antibody, a tyrosine kinase inhibitor, and an ALK inhibitor.37. The method of claim 36, wherein the tyrosine kinase inhibitor isaxitinib or palbociclib.
 38. The method of claim 36, wherein the ALKinhibitor is sunitinib or crizotinib.
 39. A method for treating cancerin a subject in need thereof, the method comprising administering to thesubject (1) an effective amount of the anti-PD-1 antibody of claim 1,and (2) an effective amount of a vaccine capable of eliciting an immuneresponse against cells of the cancer.
 40. A method for enhancing theimmunogenicity or therapeutic effect of a vaccine administered to asubject for the treatment of cancer, the method comprising administeringto the subject receiving the vaccine an effective amount of theanti-PD-1 antibody of claim
 1. 41. The method of claim 39, wherein thecancer is selected from the group consisting of breast cancer, gastriccancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer,prostate cancer, and colorectal cancer.
 42. The method of claim 40,wherein the cancer is selected from the group consisting of breastcancer, gastric cancer, liver cancer, lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, and colorectal cancer.
 43. Themethod of claim 39, further comprising administering to the subject aneffective amount of one or more other immune modulators.
 44. The methodof claim 40, further comprising administering to the subject aneffective amount of one or more other immune modulators.
 45. The methodof claim 43, wherein the other immune modulators are selected from thegroup consisting of protein kinase receptor inhibitor, a CTLA-4antagonist, a CD40 agonist, and a TLR9 agonist.
 46. The method of claim44, wherein the other immune modulators are selected from the groupconsisting of protein kinase receptor inhibitor, a CTLA-4 antagonist, aCD40 agonist, and a TLR9 agonist.